DNA Fragmentation on HCT-116 Colon Cancer Cell in the Presence of Short Chain Fatty Acid (SCFA) Extract

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DNA FRAGMENTATION ON HCT-116 COLON CANCER CELL IN THE

PRESENCE OF SHORT CHAIN FATTY ACID (SCFA) EXTRACT

Nurul Kurnia Sari and Maggy T Suhartono

Department of Food Science and Technology, Faculty of Agricultural Engineering, Bogor Agricultural University, IPB Darmaga Campus,

PO Box 220, Bogor, West Java, Indonesia

Phone: +62 858 8385 3225, e-mail: nurulkurniasari@gmail.com

ABSTRACT

Type 3 resistant starch (RS3) is retrogaded starch that resists digestion in the small intestine of healthy people and fermented in the large intestine. The main fermentation products are the short chain fatty acids (SCFA) acetate, propionate, and butyrate. Butyrate is the major energy source for the colonocytes and have anti-carcinogenic effects mainly by affecting proliferation, differentiation, and apoptosis of colonocytes. The aim of this study was to analyze apoptotic DNA fragments of HCT-116 human colon cancer cell incubated with SCFA derived from fermentation of RS3 of sweet potato (Ipomoea batatas) by C. Butyricum BCC B2571 using DNA ladder assay. The DNA bands detected from attached cells treated with SCFA containing 1.00 and 1.25 mM butyrate were different from the control, appeared as longer smear. There were separate DNA bands in the floating cells in the treated medium (SCFA containing 0.625, 1.00, and 1.25 mM butyrate) at ±100 bp. These bands were not presence in the control. DNA fragmentation was not clear in the attached cells, but was observed as a separate bands in the floating cells in the medium which contained the dead cells. This research still needs optimazion of DNA extraction to get the best result of DNA fragmentation assay.


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I.

INTRODUCTION

A.

BACKGROUND

Sweet potato (Ipomoea batatas) is an alternative food rich in starch. In Indonesia, economical value of sweet potato is low, compared with other starch sources. However, the sweet potatoes which are rich in starch made it a potential source of resistant starch. At present, various studies about resistant starch had been conducted. Recent studies showed that resistant starch consumption may reduce colorectal cancer risk.

Resistant starch has been defined as total amount of starch, and the products of starch degradation that resists digestion in the small intestine of healthy people. There are four types of resistant starch. Type 1 resistant starch (RS1) is starch that escapes digestion in the small intestine due to physical protection by the food matrix, such as grains. Type 2 resistant starch (RS2) is raw starch granules (ungelatinized) with compact structure which limits accessibility of digestive enzymes. Type 3 resistant starch (RS3) is retrogaded starch in which parts of the starch chains can crystallize into components that are less digestible. Most often, this occurs by cooking and cooling starch-containing foods. And the last, type 4 resistant starch (RS4) is not found naturally in foods. Starch that has been chemically modified to introduce bonds that are not digestible by human enzymes.

Starches that resist small intestinal breakdown are fermented by the resident bacteria in the large intestine, producing a variety of end products, the most significant of which is the short chain fatty acids (SCFA). SCFA, especially butyrate maintenance of colonic health and barrier function, butyrate has drawn most attention as this fatty acid is the major energy source for the colonocytes (Roediger 1990). Furthermore, butyrate has been shown to have anti-carcinogenic effects mainly by affecting proliferation, differentiation and apoptosis of colonocytes (Scheppach, et. al 1995).

There are several bacterial species which produce butyric acid. It is an anaerobic process. The genera Clostridium, Butyribacterium, and Butyrivibrio are the mostly studied microorganism. For commercial purposes, Clostridium species are preferred for butyric acid production. C. butyricum, C. tyobutyricum, C. thermobutyricum, C. beijerinckii, and C. populeti are of interest for the production of butyrate. Purwani, et. al (2012) observed that RS3 derived from sago and rice starch could be well utilized as substrate by Clostridium butyricum, and revealed that good proportion of acetate: propionat: butyrate in that fermentation.

Apoptosis occurs normally during development and aging and as a homeostatic mechanism to maintain cell populations in tissues and the cell is an active participant in its own demise (“cellular suicide”). Apoptosis is a form of programmed cell death characterized by cytoplasmic condensation, plasma membrane blebbing and nuclear pycnosis, leading to nuclear DNA breakdown into multiples of ~200 bp oligonucleosomal size fragments (Chen and Ioannou 1996).

One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result


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in the appearance of “DNA laddering” when the DNA is analyzed by agarose gel electrophoresis (Arends, et. al 1990). Principle of this assay is apoptotic DNA binds quickly to glass fiber fleece in the presence of a chaotropic salt, guanidine hydrochloride (guanidine HCl). After cellular impurities are washed off the fleece, the DNA is released from the fleece with a low salt buffer (Wyllie, et. al 1998).

B.

OBJECTIVE

The objective of this research was to analyze apoptotic DNA fragments of HCT-116 human colon cancer cell incubated with cell free supernatant contained short chain fatty acids (SCFA) derived from fermentation of type 3 resistant starch (RS3) of sweet potato (Ipomoea batatas) by C. Butyricum BCC B2571 using DNA ladder assay.


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II.

LITERATURE REVIEW

A.

RESISTANT STARCH (RS)

Resistant starch (RS) has been defined as total amount of starch, and the products of starch degradation that resists digestion in the small intestine of healthy people then enter the large bowel. It occurs for various reasons including chemical structure, cooking of food, chemical modification, and food mastication (Topping and Clifton 2001).There are four types of resistant starch. Starch found in plant cell walls that is inaccessible to amylase activity is one type of resistant starch, designated type 1 resistant starch (RS1). Food sources of RS1 include partially milled grains and seeds. Resistant starch also may be formed during food processing. Ungelatinized granules of starch are typically resistant to enzymatic digestion and are designated type 2 resistant starch (RS2). This type of starch can be found in potatoes and unripe (green) bananas. Cooking and cooling starchy foods by moist heat or extrude starchy foods, for example, generates retrogade starch called type 3 resistant starch (RS3). Chemical modifications of starch, such as formation of starch esters, or cross-bonded starches, also result in resistant starch, called type 4 resistant starch (RS4). RS3 and RS4 may be partially fermented by colonic bacteria (Gropper, et. al 2009). Interestingly, if RS2 granules are heated to over 100ºC, the granularity is lost and starch is gelatinized or the granules swell thereby increasing the availability of the starch to amylase. However, when the starch cools, there is some recrystallization of the starch. This is called retrogradation, which is resistant to α–amylase hydrolysis (Medeiros and Wildman 2012).

Starch that is trapped within whole plant cells or within the food matrix, and some starch granules that have not been fully gelatinized, are hydrolysed only very slowly by a-amylase and therefore may escape complete digestion in the small intestine. The energy yield to the body from this source is less than that provided if starch is digested and absorbed in the small intestine (Englyst, et. al 1996). The glycemic effects of starch is therefore lowered, which decreases insulin secretion. In addition to glycemic management resistant starches may play role in weight management as well (Medeiros and Wildman 2012).

The material included in the definition of RS reaches the human large intestine and thus becomes a substrate for microbial fermentation. The end-products are H2and CO2, CH4 in about half the population, and short-chain fatty acids. SCFA consist primarily of butyrate, propionate and acetate (Englyst, et. al 1996). SCFA contribute to normal large bowel function and prevent pathology through their actions in the lumen and on the colonic musculature and vasculature, through their metabolism by colonocytes (Topping and Clifton 2001).

B.

SHORT CHAIN FATTY ACID (SCFA)

Short chain fatty acids (SCFA) are end-product of luminal microbial fermentation of predominantly non-digestible dietary carbohydrates. SCFA with different carbon chain lengths (acetate (C2), propionate (C3), butyrate (C4), valerate (C5), and caproate (C6)) are produced in


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varying amounts depending on the diet and the composition of the intestinal microbiota (Marleen,

et. al 2003). SCFA constitute approximately two-thirds of the colonic anion concentration (70-130 mmol/l), mainly as acetate, propionate, and butyrate (Mortensen and Clausen 1996). Various population data show that SCFA production is in order of acetate > propionate > butyrate in a molar ratio of approximately 60:20:20 or 3:1:1, respectively in the proximal and distal colon (Cummings, et. al 1987; Topping and Clifton 2001).

The major source of this in the human colon is thought to be plant cellwall polysaccharides such as cellulose, pectins, and hemicelluloses, currently referred to in human nutrition as dietary fibre. Starch would also be a suitable substrate if it were to reach the colon in significant quantities. Plant cell-wall polysaccharides are composed of hexose (glucose and galactose), pentose (xylose and arabinose), and uronic acid monomers which are fermented by gut micro-organisms along a variety of pathways. The important feature to remember of this metabolism is that it is anaerobic. Many lines of evidence therefore support the idea that fermentation occurs in the colon but, because of the difficulties in obtaining access to the human large bowel during normal digestion, most of the evidence for fermentation has, of necessity, been indirect (Cummings, et. al 1987).

There are several bacterial species which produce butyric acid. Most butyric acid-producing bacteria form acetic acid in addition to butyric acid as their major fermentation products.

Clostridium butyricum, C. tyobutyricum, C. thermobutyricum, C. beijerinckii, and C. populeti are of interest for the production of butyrate. There are some benefits using Clostridium butyricum.

These bacteria needs simple growth medium, produces high metabolite, and easy to isolate. Therefore, Clostrium butyricum also has butyrogenic activity, produce more butyric acid than acetic acid and propionic acid. C. butyricum had the highest specific growth rates compared with other genera. On the other hand, Clostridium spp. are generally less dominant in the human colon and hence the role of Clostridium in the production of butyrate in humans is unclear (Wang 1999).

SCFA will be absorbed and contribute about 3 kilocalories per gram (Medeiros and Wildman 2012). SCFA are rapidly absorbed and have shown to have distinct bioactivity depending on their chain length. With regard to maintenance of colonic health and barrier function, butyrate has drawn most attention as this fatty acid is the major energy source for the colonocytes (Roedriger 1990). Furthermore, butyrate has been shown to have anti-carcinogenic effects mainly by affecting proliferation, differentiation, and gene expression of colonocytes (Scheppach, et. al 1995). Butyrate concentration didn‟t show a significant correlation with pH or

propionic concentration (McIntyre, et. al 1993). The production of propionate is by two main, but circuitous, routes, firstly, involving fixation of CO2, to form succinate which is subsecquently decarboxylated (the 'decarboxylic acid pathway') or secondlly, from lactate and acrylate (the 'acrylate pathway'). Acetate is usually formed by the oxidative decarboxylation of pyruvate and butyrate by reluction of acetoacetate formed from acetate (Cummings, et. al 1981).

Short chain fatty acids are metabolized at 3 major sites in the body (Hijova and Chmelarova 2007):


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1. Cells of the ceco-colonic epithelium that use butyrate as a major substrate for the maintenance of energy producing path-ways;

2. Liver cells that metabolize residual butyrate with propionate used for gluconeogenesis; 50% to 70% of acetate is also taken up by the liver;

3. Muscle cells that generate energy from the oxidation of residual acetate.

In ruminants and other herbivores, SCFA are absorbed and transported via the portal vein to the liver, and the fraction not absorbed is distributed to the other body organs and tissues for metabolism. In herbivores, peripheral venous SCFA concentrations are high due to comparatively low visceral extraction and high rates of absorption into the circulation. However, human peripheral venous blood concentrations are normally low, and only acetate is present in measurable amounts. This profile reflects the lower SCFA production rates and greater visceral extraction in omnivores, meaning that human peripheral venous SCFA are not representative of those in the portal circulation. Human experimentation has been confined largely to fecal measurements, which are also limited as 95% of SCFA are produced and absorbed within the colon (Topping and Clifton 2001).

Purwani, et. al (2012) study revealed that type 3 resistant starch (RS3) derived from sago and rice starch that treated with pullulanase could be well utilized as substrate by Clostridium butyricum and showed good proportion of acetate: propionate: butyrate in the bacterial culture filtrate. The fermentation of resistant starch type 3 (RS3) derived from sago starch treated with pullulanase (RSSP) by C. butyricum produced 17.65 mM of butyrate, and fermentation of RS3 derived from rice starch treated with pullulanase (RSRP) by C. butyricum produced 21.76 mM, higher concentration than fermentation of RSSP. This study implied that difference of starch source resulted difference of SCFA concentration.

C.

APOPTOSIS

Apoptosis (“normal” or “programmed” cell death) is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Apoptosis is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise (“cellular suicide”) (Wyllie, et. al 1998). Apoptosis is a form of programmed cell death characterized by cytoplasmic condensation, plasma membrane blebbing and nuclear pycnosis, leading to nuclear DNA breakdown into multiples of ~200 bp oligonucleosomal size fragments (Chen and Ioannou 1996).


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Figure 1 Illustration of the morphological features of apoptosis (Wyllie, et. al 1998)

Apoptosis is characterised by an initial shrinkage of the cell, which in a tissue means breaking cell-to-cell contacts with neighbours and rounding up. The consequence of this stage tends to be that the volume of the cell becomes smaller, and the cytoplasmic internal membranes, ribosomes, mitochondria, and other cytoplasmic organelles are more concentrated in the cytoplasm, which then consequently looks darker. The organelles remain intact and healthy looking very late into the process of death, suggesting that the cell continues metabolic activity for some considerable time. This indeed can be demonstrated in many cell systems by using various inhibitors of metabolic processes (RNA and protein synthesis inhibitors), which delay the progression of a cell through the apoptotic sequence. Apoptosis characteristically involves single isolated cells and not clusters. The DNA or chromatin material in the nucleus condenses very extreme (Potten and Wilson 2004). Pyknosis is the result of chromatin condensation and this is the most characteristic feature of apoptosis. On histologic examination with hematoxylin and eosin stain, apoptosis involves single cells or small clusters of cells. The apoptotic cell appears as a round or oval mass with dark eosinophilic cytoplasm and dense purple nuclear chromatin fragments. Electron microscopy can better define the subcellular changes. Early during the chromatin condensation phase, the electron-dense nuclear material characteristically aggregates peripherally under the nuclear membrane although there can also be uniformly dense nuclei.

Extensive plasma membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies during a process called “budding.” Apoptotic bodies consist of cytoplasm with tightly packed organelles with or without a nuclear fragment. The organelle integrity is still maintained and all of this is enclosed within an intact plasma membrane. These bodies are subsequently phagocytosed by macrophages, parenchymal cells, or neoplastic cells and degraded within phagolysosomes. Macrophages that engulf and digest apoptotic cells are called “tingible body macrophages” and are frequently found within the reactive germinal centers of lymphoid follicles or occasionally within the thymic cortex. The tingible bodies are the bits of nuclear debris from the apoptotic cells. There is essentially no inflammatory reaction associated with the process of apoptosis nor with the removal of apoptotic cells because: (1) apoptotic cells


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do not release their cellular constituents into the surrounding interstitial tissue; (2) they are quickly phagocytosed by surrounding cells thus likely preventing secondary necrosis; and (3) the engulfing cells do not produce anti-inflammatory cytokines (Savill and Fadok 2000; Kurosaka, et. al 2003).

The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, an irreversible event that commits the cell to die and occurs before changes in plasma membrane permeability (prelytic DNA fragmentation). The fragmentation of chromosomal DNA is a hallmark of apoptosis and may facilitate apoptosis by terminating DNA replication and gene transcription. In many systems, this DNA fragmentation has been shown to result from activation of an endogenous Ca2+ and Mg2+ dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites located between nucleosomal units (linker DNA) generating mono- and oligonucleosomal DNA fragments (Arends et. al, 1990; Wyllie, et. al 1998).

In priciple, there are two main apoptotic pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. In both pathways, cysteine aspartyl-specific proteases (caspases) are activated that cleave cellular substrates, and leads to the biochemical and morphological changes that are characteristic of apoptosis. However, there is now evidence that the two pathways are linked and that molecules in one pathway can influence the other (Igney and Krammer 2002). Figure 2 shows the two main apoptotic signalling pathways.


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Apoptotic cells exhibit several biochemical modifications such as protein cleavage, protein cross-linking, DNA breakdown, and phagocytic recognition that together result in the distinctive structural pathology described previously (Hengartner 2000). Caspases are widely expressed in an inactive proenzyme form in most cells and once activated can often activate other procaspases, allowing initiation of a protease cascade. Some procaspases can also aggregate and autoactivate. This proteolytic cascade, in which one caspase can activate other caspases, amplifies the apoptotic signaling pathway and thus leads to rapid cell death (Elmore 2007).

Although DNA fragmentation into oligonucleosomal ladders is characteristic of apoptosis, recent evidence indicates that not all cells undergo such extensive DNA fragmentation (Cohen, et. al1. 1992 in Chen and Ioannou 1996). In fact, fragmentation of DNA into kilobase-size fragments appears to be an early event in apoptosis, preceding the complete digestion of DNA into multiples of nucleosomal size fragments (Cohen and Sun 1994 in Chen and Ioannou 1996).

D.

DNA LADDER ASSAY

One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of “DNA laddering” when the DNA is analyzed by agarose gel electrophoresis (Arends, Morris, and Wyllie 1990). Principle of this assay is apoptotic DNA binds quickly to glass fiber fleece in the presence of a chaotropic salt, guanidine hydrochloride (guanidine HCl). After cellular impurities are washed off the fleece, the DNA is released from the fleece with a low salt buffer (Wyllie, et. al 1998).

DNA ladder is asolution of DNA molecules of different lengths used in agarose gel electrophoresis. it is applied to an agarose gel as a reference to estimate the size of unknown DNA molecules. In addition it can be used to approximate the mass off a band by comparison to a special mass ladder. Different DNA ladders are commercially available depending on expected DNA length. The 1 kb ladder with fragment ranging from about 0.5 kbp to 10 or 12 kbp and the 100 bp ladder with fragment ranging from 100 bp to just above 1000 bp are the most frequent. DNA ladders are often produced by a suitable restriction digest of plasmid.

DNA electrophoresis is an analytical technique used to separate DNA fragments by size. An electric field forces the fragments to migrate through a gel. DNA molecules normally migrate from negative to positive potential due to the net negative potential due to the net negative charge of the phosphate backbone of the DNA chain. At the scale of the length of DNA molecules, the gel looks much like a random, intricate network. Longer molecules migrate more slowly because they are more easily „trapped‟ in the network. After the separation is completed, the fractions of DNA fragments of different length are often visualized using a fluorescent dye spesific for DNA, such as ethidium bromide. The gel shows bands corresponding to different DNA molecules populations with different molecular weight. Fragment size is usually reported in “nucleotides”, “base pairs” or


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separated. Fragment size determination is typically done by comparison to commercially available DNA ladders containing linear DNA fragments of known length.

The types of gel most commonly used for DNA electrophoresis are agarose (for relatively long DNA molecules) and polyacrylamide (for high resolution of short DNA molecules, for example in DNA sequencing). Electrophoresis techniques used in the assessment of DNA damage include alkaline gel electrophoresis and pulsed field gel elctrophoresis. The measurement and analysis are mostly done with a specialized gel analysis software.

DNA molecules must be separated from other cellular material before they can be examined. Cellular proteins that package and protect DNA in the environment of the cell can inhibit the ability to analyze the DNA. Therefore, DNA extraction methods have been developed to separate proteins and other cellular materials from the DNA molecules. The quantity and quality of DNA often need to be measured prior to proceeding further with analytical procedures to ensure optimal results. Organic extraction was for many years the most widely used methods for DNA extraction. Organic extraction involves the serial addition of several chemicals. First sodium dodecylsulfate (SDS) and proteinase K are added to break open the cell walls and to break down the proteins that protect the DNA molecules while they are in chromosomes (Butler 2010). NA sample purity and molecular weight must be controlled by agarose gel electrophoresis with 0.4-0.8% ethidium bromide stain. The DNA can be suspended in TE buffer (Tris-HCL and EDTA pH 8.0) or double-distilled water (Walker and Rapley 2008).


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III.

MATERIALS AND METHODS

A.

MATERIALS AND EQUIPMENTS

1. MATERIALS

The sample used for this research was the fermentation product of type III resistant starch (RS3) derived from Ipomoea batatas var. Jago provided by Indonesian Center of Agricultural Post Harvest Research and Development, Bogor in the form of cell free supernatant consisted of 52.95 mM acetic acid, 66.93 mM propionic acid, and 92.41 mM butyric acid. HCT-116 colon cancer cells was provided by Primate Research Center, Bogor Agricultural University. Materials used in this research were Dulbecco‟s Modified Eagle‟s Medium (DMEM) Gipco USA, Fetal Bovine Serum (FBS) Gipco USA, antibiotics (penicillin and streptomycin) Invitrogen USA, Phosphate Buffered Saline (PBS) Gipco USA, Trypsin 0.25% Difco USA, Qiagen DNA extraction kit (lysis buffer, Aw 1, Aw 2, and AE buffer), agarose powder Promega, ethidium bromide Gipco USA, gel loading buffer, double-distilled water, and TAE buffer 50x Gipco USA (0.484 gram Tris-Base, 0.1142 mL acetic acid, and 0.2 mL 0.5M EDTA pH 8.0).

2. EQUIPMENT

The equipments used in this research were biosafety cabinet, dissposable gloves, dissposable pipettes (1 mL, 2 mL, 5 mL, 10 mL, 25 mL, and 50 mL), micropipette, T25 and T75 tissue culture flask, incubator (37oC, 5% CO2), refrigerator, water-bath, inverted microscope, centrifuge, centrifuge tube, glass bottle, gel doc, cuvette, and DNA electrophoresis.

B.

METHODS

1. Cell Culture

HCT-116 colon cancer cells were warmed up from cryogenic state using waterbath with 37 ºC. Then, the cells were pelleted using centrifuge 750 g and media was discarded. HCT-116 colon cancer cells have been cultured in Dulbecco‟s Modified Eagle‟s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics (penicillin 100 unit/mL; streptomycin 100µg/mL). Cells (1 x 105) were seeded into T25 tissue culture flask. The cells were incubated at 37oC in 5% CO2 incubator.

The growth medium was changed every 2-3 days.The cells were subcultured every week or when the cells already reach 100% confluency. Subculture consisted of several steps. First, the medium were discarded and the cells were washed using Phosphate Buffered Saline (PBS) to make sure that there wasn‟t any media left in the flask. Next, the cells were trypsinized with Trypsin 0,25% to free the cells from adhesion to the flask followed by incubation in 37 ºC for 5-10 minutes. After that, the sample was centrifuged 750 g for 5 minutes used to pellet the cells, and the supernatant was discarded. The pellet were resuspended using fresh medium and counted using


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hemocytometer to seed the cell into flasks in an acurate amount. 1 × 105 cells were seeded into T25 flask every subculture for maintaining.

2. Incubation with SCFA

Analysis was done using eight T25 flasks which contained 1 x 105 cells as the starting cell number. The flasks was incubated for 2 x 24 hr or to reach 50% confluency. Next, incubation was continued for another 4 x 24 hr in the absence (control) or presence of various concentration of cell free supernatant contained SCFA (0.625 mM, 1.00 mM, and 1.25 mM butyrate in the cell free supernatant). For the treatment of the experimental cultures, the cell free supernatant contained SCFA was diluted with DMEM. At the end of 4 x 24 hr incubation period, the cells were observed using inverted microscope with magnification (4x10). Then, the medium contained of dead cells was collected into centrifuge tube and the attached cells were washed with phosphate buffered saline (PBS). The medium contained of dead cells that floating in the medium and made the medium became turbid. After that, the attached cells were trypsinized with trypsin 0.25% to free the cells from adhesion to the flask followed by incubation in 37 ºC for 5-10 minutes and pipetted into centrifuge tube. Next, the attached cells in the trypsin and the medium were pelleted with centrifugation 750 g for 5 minutes. After that, the supernatant was discarded. The pellet were resuspended using 10 mL of PBS steril and centrifuged as before. Then, the supernatant was discarded. The pellet were resuspended using 1 mL PBS steril, then pipetted into eppendorf. Next, attached and dead cells were centrifuged 1000 g for 5 menit. The supernatant was discarded. And the last, the cuvettes were stored at -20oC.

3. DNA Extraction (Qiagen DNA Extraction Kit)

The cells pellet were treated for 10 minutes with 200 μl lysis buffer. Then, the sample was mixed with ethanol 95% and pipetted into filter tube (silica gel). The filter tube was centrifuged 8,000 g for 1 minute and the flow through was discarded. After that, the treated and untreated cells were washed with wash buffer (Aw 1) and centrifuged as before. Next, the treated and untreated cells were washed with wash buffer (Aw 2) and centrifuged 8,000 rpm for 3 minutes. Then, the sample was mixed with 100 μL elution buffer (AE buffer) and centrifuged 8,000 rpm for 1 minute. 4. DNA Ladder Assay

Eluted DNA was mixed with gel loading buffer and applied to 1% agarose gel which contained ethidium bromide. Agarose gel was made from 1 gram agarose powder, 98 mL double-distilled water, and 2 mL buffer TAE (0.484 gram Tris-Base, 0.1142 mL acetic acid, and 0.2 mL 0.5M EDTA pH 8.0). Next, the gel was run in TAE (Tris-Acetate EDTA) buffer at 100 V for 1 hours. And the last step was visualization of DNA ladder pattern using gel doc.


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IV.

RESULTS AND DISCUSSION

A.

INHIBITION TO HCT-116 COLON CANCER CELL

After 4 x 24 hr incubation in the presence and absence of the cell free supernatant contained SCFA, observation was conducted using inverted microscope with magnification (4x10). Cellular debris in the media could be observed on HCT-116 cells that were treated with SCFA containing 0.625, 1.00, and 1.25 mM butyrate (figure 3). This research showed that the attached cells of control became multi layer. The treated cells with 1.00 mM butyrate showed higher confluency than the treated cells with 1.25 mM butyrate, but lower than the treated cells with 0.625 mM butyrate. This results implied that higher concentration of butyrate showed higher inhibition to HCT-116 colon cancer cell. The inhibition effect was implied at butyrate concentration as low as 0.625 mM and was lower than those reported by Purwani, et. al (2010) that the inhibition effect was observed at butyrate concentration as low as 2.6 mM. SCFA derived from fermentation of RS3 of sago decreased the viability, the total cell number, and increased the inhibition growth of HCT-116 colon cancer cell in a dose dependent manner (p>0.05).

Other research concluded that butyrate inhibited Caco-2 cell proliferation at concentration as low as 0.1 mM (Ruemmele, et. al 1999). This research implied inhibition at higher concentration of butyrate. This difference might caused by cancer cell difference that was used in each research, therefore difference in effect may occur.

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Figure 3 Morphological change of untreated or treated HCT-116 with cell free supernatant contained SCFA. (1) untreated cells; (2) cells treated with 0.625 mM; (3) cells treated with 1.00 mM; (4) cells treated with 1.25 mM


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B.

DETECTION OF APOPTOSIS USING DNA LADDER ASSAY

DNA fragmentation of HCT-116 colon cancer cell was observed with DNA ladder assay using electrophoresis gel. Observartion data showed different DNA bands between control and cells treated with SCFA (figure 4). DNA bands from attached cells treated with SCFA containing 0.625 mM butyrate was not significantly different from the control. However, the attached cells treated with SCFA containing 1.00 and 1.25 mM butyrate had DNA bands that were significantly different from the control: appeared as longer smear.

This research also observed DNA fragmentation of the floating cells in the medium. There were different result of DNA bands of the floating cells in the control medium and in the treated medium. There were thin DNA bands in the floating cells in the treated medium (SCFA containing 0.625, 1.00, and 1.25 mM butyrate) at ±100 bp (compared with commercial ladder), lower density than normal DNA bands. These bands were not presence in the control. This result showed that DNA of floating cells in the treated medium was fragmented and had lower density than control, the bands observed at 180-200 bp.

The appearance of the nucleosomal DNA ladder in agarose gels has become the hallmark of programmed cell death via apoptosis. This research implied that SCFA derived from fermentation of type-3 resistant starch of Ipomoea batatas induced apoptosis via DNA fragmentation. DNA fragmentation was not clear in the pellet of attached cells, but was observed as a separate bands in the pellet of floating cells in the medium. Floating cells in the medium contained dead cells. This imply that SCFA induced apoptosis at very early stage in the attached cells and later stage in the floating cells in the medium which contained of dead cells.

Other researchers had reported that butyrate inhibition towards cancer cells are via apoptosis (Purwani and Suhartono 2010; Ruemmele, et. al 1999; Hinnebusch, et. al 2002). Many studies investigated the mechanism of how SCFA induced apoptosis. Purwani and Suhartono (2010) studies showed that SCFA in bacterial supernatant induced apoptosis and changed the expression of apoptosis related genes such as Bcl-2 and Bax. It also showed that bacterial supernatant consisted of SCFA increased the expression ratio of pro-apoptotic genes.

Other research concluded that SCFA especially butyrate effects are mainly due to its regulation of gene expression. That research simultaneously assessed the expression levels of 19,400 human genes in response to butyrate in the human colonic epithelial cell line, HT29, and found that over 10% were differentially expressed (compared to the controls) (Daly and Shirazi 2006). Studies using HeLa cells which have been exposed to sodium butyrate showed the level of histone acetylation was greatly increased as compared to control cells. The experiments indicate that butyrate inhibited activity of histone deacetylase, which influenced of the chromatin structure of the nucleosomal level (Bofa, et. al 1978).


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Figure 4 DNA fragmentation of untreated and treated HCT-116 colon cancer cells

Lane Identification

1 : Floating cells in the control medium

2 : Floating cells in the treated medium with SCFA containing 0.625 mM butyrate

3 : Floating cells in the treated medium with SCFA containing 1.00 mM butyrate

4 : Floating cells in the treated medium with SCFA containing 1.25 mM butyrate

5 : Attached cells control

6 : Attached cells treated with SCFA containing 0.625 mM butyrate 7 : Attached cells treated with SCFA

containing 1.00 mM butyrate 8 : Attached cells treated with SCFA

containing 1.25 mM L : Ladder 100 bp


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Hinnebusch, et. al (2002) treated HT-26 and HCT-116 colon cancer cell with physiologically relevant concentrations of various SCFA, and apoptotic effects were studied by flow cytometry. Butyrate significantly increased apoptosis, whereas the other SCFA studied did not. Hinnebusch, et. al (2002) concluded that SCFA effects on apoptosis, and interestingly, only C4 (butyrate) increased the rate of programmed cell death.

Rummele, et. al (1999) studies showed that butyrate induced apoptosis (maximum 79%) Caco-2 cell line via activation of the caspase-cascade (a key event was the proteolytic activation of caspase-3, triggering degradation of poly-(ADP-ribose) polymerase (PARP)) and the mitochondrial bcl-pathway (butyrate potently up-regulatef the expression of the pro-apoptotic protein bak, without changing Caco-2 cell bcl-2 expression). Bak is a protein that induce pore formation on mitochondrial surface, therefore releasing cytochrome-c out of mitochondrial membrane forming apoptosome that could lead to activation of caspase cascade which could induce apoptosis. The caspase play an important role in apoptosis by activating Dnases, inhibiting DNA repair enzymes and breaking down structural protein in the nucleus (Elmore 2007). In summary, apoptosis is more likely when bax protein levels are high (Wyllie, et. al 1998).

The expected result of this study was clear DNA fragmentation, and there were differences of DNA fragmentation of normal and apoptosis cells. However, DNA fragmentation in this research was not clearly observed. Eventhough, variation of the concentration of agarose gel (0.8%, 1%, and 2%), voltage (75 and 100 Volt), and electrophoresis time (1 and 1.5 hours) was treated, the expected DNA fragmentation was not seen clearly. This might be related with DNA extraction methods that was not optimal, and thus affected the DNA bands in the agarose.

Figure 5. Comparison of two methods for the preparation of apoptotic DNA fragments (Herrmann, et. al 1994) Lane identification:

M : 142 bp ladder

1 : phenol extraction method, 0 hr propidium iodide staining, 2 : phenol extraction method, 3 hr propidium iodide staining 3 : phenol extraction method, 14 hr propidium iodide staining 4 : NP-40 lysis extraction method, 0 hr propidium iodide staining 5 : NP-40 lysis extraction method, 3 hr propidium iodide staining 6 : NP-40 lysis extraction method, 14 hr propidium iodide staining


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16

Herrmann, et. al (1994) compared the methods for isolation of apoptotic DNA fragments. The research showed that method for isolation of apoptotic DNA affected the bands of DNA fragmentation. Figure 5 shows that the NP-40 lysis method with a frequently used method for isolation of apoptotic. It shows that when NP-40 lysis method produces better DNA fragmentation (figure 5 lane 4-7) compares with phenol extraction procedures (figure 5 lane 1-3). This research used ethanol extraction procedure. The fragmentation of DNA in this research was similar not clear. This research still needs optimazion methodology of DNA extraction to get the best result of DNA fragmentation assay.


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V.

CONCLUSION AND RECOMMENDATION

A.

CONCLUSION

Based on this research, Short Chain Fatty Acids(SCFA) derived from fermentation of type-3 resistant starch of Ipomoea batatas containing 1,00 and 1, 25 mM butyrate produced DNA bands different from the control when measured with DNA ladder assay. The DNA bands of the treated cells appeared as longer smear. There were separate DNA bands in the floating cells in the treated medium (SCFA containing 0.625, 1.00, and 1.25 mM butyrate) at ±100 bp. These bands were not presence in the control. DNA fragmentation was not clear in the attached cells, but was observed as a separate bands in the floating cells in the medium. This imply that SCFA might induce apoptosis at very early stage in the attached cells and later stage in the floating cells in the medium.

B.

RECOMMENDATION

DNA fragmentation in this research was not clearly/optimally observed. This might relate to suboptimal DNA extraction. Isolation of apoptotic cells and optimazion methodology of DNA extraction should be done to get the best result of DNA fragmentation assay.


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DNA FRAGMENTATION ON HCT-116 COLON CANCER CELL

IN THE PRESENCE OF SHORT CHAIN FATTY ACID (SCFA)

EXTRACT

BACHELOR THESIS

NURUL KURNIA SARI

F24080056

FOOD SCIENCE AND TECHNOLOGY

FACULTY OF AGRICULTURAL ENGINEERING

BOGOR AGRICULTURAL UNIVERSITY


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DNA FRAGMENTATION ON HCT-116 COLON CANCER CELL

IN THE PRESENCE OF SHORT CHAIN FATTY ACID (SCFA)

EXTRACT

BACHELOR THESIS

NURUL KURNIA SARI

F24080056

FOOD SCIENCE AND TECHNOLOGY

FACULTY OF AGRICULTURAL ENGINEERING

BOGOR AGRICULTURAL UNIVERSITY


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DNA FRAGMENTATION ON HCT-116 COLON CANCER CELL IN THE

PRESENCE OF SHORT CHAIN FATTY ACID (SCFA) EXTRACT

Nurul Kurnia Sari and Maggy T Suhartono

Department of Food Science and Technology, Faculty of Agricultural Engineering, Bogor Agricultural University, IPB Darmaga Campus,

PO Box 220, Bogor, West Java, Indonesia

Phone: +62 858 8385 3225, e-mail: nurulkurniasari@gmail.com

ABSTRACT

Type 3 resistant starch (RS3) is retrogaded starch that resists digestion in the small intestine of healthy people and fermented in the large intestine. The main fermentation products are the short chain fatty acids (SCFA) acetate, propionate, and butyrate. Butyrate is the major energy source for the colonocytes and have anti-carcinogenic effects mainly by affecting proliferation, differentiation, and apoptosis of colonocytes. The aim of this study was to analyze apoptotic DNA fragments of HCT-116 human colon cancer cell incubated with SCFA derived from fermentation of RS3 of sweet potato (Ipomoea batatas) by C. Butyricum BCC B2571 using DNA ladder assay. The DNA bands detected from attached cells treated with SCFA containing 1.00 and 1.25 mM butyrate were different from the control, appeared as longer smear. There were separate DNA bands in the floating cells in the treated medium (SCFA containing 0.625, 1.00, and 1.25 mM butyrate) at ±100 bp. These bands were not presence in the control. DNA fragmentation was not clear in the attached cells, but was observed as a separate bands in the floating cells in the medium which contained the dead cells. This research still needs optimazion of DNA extraction to get the best result of DNA fragmentation assay.


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Nurul Kurnia Sari. F24080056. DNA Fragmentation on HCT-116 Colon Cancer Cell in the Presence of Short Chain Fatty Acid (SCFA) Extract. Supervised by Maggy Thenawidjaja Suhartono 2012.

SUMMARY

Sweet potato (Ipomoea batatas) is an alternative food rich in starch, made it a potential source of resistant starch. Recent studies showed that resistant starch consumption may reduce colorectal cancer risk. Resistant starch is total amount of starch or the products of starch degradation that resists digestion in the small intestine of healthy people. There are four types of resistant starch.

Type 3 resistant starch (RS 3) is retrogaded starch, may be partially fermented by colonic bacteria. The end-products are H2, CO2, CH4, and short chain fatty acid (SCFA). SCFA consists primarily of butyrate, propionate, and acetate. SCFA, especially butyrate maintenance of colonic health and barrier function, butyrate has drawn most attention as this fatty acid is the major energy source for the colonocytes. Furthermore, butyrate has been shown to have anti-carcinogenic effects mainly by affecting proliferation, differentiation and apoptosis of colonocytes.

The objective of this research was to analyze apoptotic DNA fragments of HCT-116 human colon cancer cell incubated with short chain fatty acids (SCFA) derived from fermentation of type 3 resistant starch (RS3) of sweet potato (Ipomoea batatas) by C. Butyricum BCC B2571 using DNA ladder assay.

The DNA bands detected from attached cells treated with SCFA containing 1.00 and 1.25 mM butyrate were different from the control, appeared as longer smear. There were separate DNA bands in the floating cells in the treated medium (SCFA containing 0.625, 1.00, and 1.25 mM butyrate) at ±100 bp. These bands were not presence in the control. DNA fragmentation was not clear in the attached cells, but was observed as a separate bands in the floating cells in the medium which contained the dead cells. This research still needs optimazion of DNA extraction to get the best result of DNA fragmentation assay.


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DNA FRAGMENTATION ON HCT-116 COLON CANCER CELL IN THE

PRESENCE OF SHORT CHAIN FATTY ACID (SCFA) EXTRACT

BACHELOR THESIS

In the partial fulfillment of the requirement of degree of

SARJANA TEKNOLOGI PERTANIAN

at the Department of Food Science and Technology

Faculty of Agricultural Engineering

Bogor Agricultural University

By :

NURUL KURNIA SARI

F24080056

FACULTY OF AGRICULTURAL ENGINEERING

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

2012


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Title

: DNA Fragmentation on HCT-116 Colon Cancer Cell in the

Presence of Short Chain Fatty Acid (SCFA) Extract

Name

: Nurul Kurnia Sari

Student ID : F24080056

Approved by,

Academic Advisor,

(Prof. Dr. Ir. Maggy Thenawidjaja Suhartono)

NIP. 19530507 197701 2 001

Acknowledged by,

Head of Department of Food Science and Technology,

(Dr. Ir. Feri Kusnandar, M. Sc)

NIP 19680526 199303 1 004


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STATEMENT LETTER OF THESIS AND SOURCE OF

INFORMATION

Hereby I genuinely stated that the bachelor thesis entitled DNA Fragmentation on HCT-116 Colon Cancer Cell in the Presence of Short Chain Fatty Acid (SCFA) Extract is an authentic work of mine under supervision of academic advisor and never being presented in any forms and universities. All the informations taken and quoted from published or unpublished works of other writers had been mentioned in the texts and attached in the references at the end of the bachelor thesis.

Bogor, July 2011 The undersigned,

Nurul Kurnia Sari F24080056


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© Created right owned by Nurul Kurnia Sari, 2012 All rights reserved

This thesis may not be translated or copied in whole or in part without written permission from Bogor Agricultural University in any forms or by any forms,print, photocopy, microfilm, etc.


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AUTHOR BIOGRAPHY

The author was born in Purworejo, on November 30th 1991 to Mulyono and Pariyah couple. She is the youngest daughter with one brother, Achmad Kurniawan. She studied in Kindergarten Tunas Gama Kaligesing (1996-1997), Elementary of Kaligesing (1997-2003), Junior High School 3 Purworejo (2003-2006), Senior High School 1 Purworejo acceleration program (2006-2008). In 2008, the author continued her further study at IPB with USMI (Undangan Seleksi Masuk IPB) path and she was accepted as Food Science and Technology student in the Faculty of Agriculture Engineering. During her study, she joined organizations such as Dormitory IPB, DPM KM IPB (Dewan Perwakilan Mahasiswa Keluarga Mahasiswa IP), MPM KM IPB (Majelis Permusyawaratan Mahasiswa Keluarga Mahasiswa IPB), GAMAPURI (Keluarga Mahasiswa Purworejo di IPB) and she was active in activities such as Loka Karya Kemahasiswaan Keluarga Mahasiswa IPB (2008), Pemira Keluarga Mahasiswa (2009), Tetranology (2009), Suksesi Himitepa (2009), Save Our Earth (2010), PLASMA (2010), and HACCP (2010). The author has work expriences as Team production of Substitusion Corn Noodles (2009-2010), Tutor Al-Fattaah (2010-2012), and General Secretary of Al-Fattaah Private Tutoring Organization in Bogor (2011-2012). The achievements that the author get was The best teacher of Al-Fattaah Private Tutoring (2010). She was a speaker at Enterpreneurial Berbasis Potensi Lokal (2012). In September 2011, she started her research entitled “DNA Fragmentation on HCT-116 Colon Cancer Cell in the Presence of Short Chain Fatty Acid (SCFA) Extract” supervised by Prof. Dr. Ir. Maggy Thenawidjaja Suhartono.


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PREFACE

First and foremost, Praise to Allah for giving me strength to accomplish my research and thesis. All Your blessing motivated my self to achieve my bachelor degree. This bachelor thesis which is entitled “DNA Fragmentation on HCT-116 Colon Cancer Cell in the Presence of Short Chain Fatty Acid (SCFA) Extract”, was based on research conduced from September 2011 to Maret 2012. The author would like to thank:

1. My father Mulyono, my mother Pariyah, and my brother Achmad Kurniawan. Thank you for all your love, support, and pray.

2. Prof. Dr. Ir. Maggy Thenawidjaja Suhartono as my academic supervisor. Thenk you for her enormous help both academically and financially throughout the completion of my thesis. I am eternally grateful both her inspiring advice, warm support and her encouragement. 3. Dr. Endang Yuli Purwani for providing all my research requirements, all her guidances, and

also as my thesis examiner.

4. Dr. Nancy Dewi Yuliana as my thesis examiner, whose critism and suggestion would help me accomplish my thesis.

5. Silmi Mariya, S.Si, Uus Saepuloh, S.Si, M.Biomed, and Ms. Sela for their guidances in the laboratory. I am grateful for their helpful suggestions and advices.

6. Lecturers of Department of Food Science and Technology for the knowledge that enable me to do this research.

7. My lovely nephew: Ayesha Ahmad, Kayysah Ahmad, and Yumna Kinasih.

8. Desy Ayu as my partner in research. Thanks for the times that we have spent together for disscussion, culinary, and etc.

9. My beloved “TACOS” member ITP45.

10. Al-Fattaah Private Tutoring member. Thank you for being my family, supporting me, make me learn more how to manage my self and how to manage people.

11. Member of Freaky: Meutia, Arum, Anita, Nanda, and Icha. Thanks for being my partner in crime, doing crazy and silly things.

12. Staff of UPT Food Science and Technology Department: Bu Novi and Mbak Anie

Bogor, Mei 2012


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TABLE OF CONTENT

Page TABLE OF CONTENT ... ix LIST OF FIGURE ... x I. INTRODUCTION ... 1 A. BACKGROUND ... 1 B. OBJECTIVE ... 2 II. LITERATURE REVIEW ... 3 A. RESISTANT STARCH (RS) ... 3 B. SHORT CHAIN FATTY ACID (SCFA) ... 3 C. APOPTOSIS ... 5 D. DNA LADDER ASSAY ... 8 III. MATERIALS AND METHODS ... 10

A. MATERIALS AND EQUIPMENTS ... 10 1. MATERIALS ... 10 2. EQUIPMENTS ... 10 B. METHODS ... 10 1. Cell Culture ... 10 2. Incubation with SCFA ... 11 3. DNA Extraction (Qiagen DNA Extraction Kit) ... 11 4. DNA Ladder Assay ... 11 IV. RESULTS AND DISSCUSSION ... 12 A. INHIBITION TO HCT-116 COLON CANCER CELL ... 12 B. DETECTION OF APOPTOSIS USING DNA LADDER ASSAY ... 13 V. CONCLUSION AND RECOMMENDATION ... 17 A. CONCLUSION ... 17 B. RECOMMENDATION ... 17 REFERENCES ... 18


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LIST OF FIGURE

Page Figure 1. Illustration of the morphological features of apoptosis ... 6 Figure 2. The two main apoptotic signalling pathways ... 7 Figure 3. Morphological change of untreated or treated HCT-116 with cell free supernatant

contain SCFA ... 12 Figure 4 DNA fragmentation of untreated and treated HCT-116 colon cancer cells 14


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1

I.

INTRODUCTION

A.

BACKGROUND

Sweet potato (Ipomoea batatas) is an alternative food rich in starch. In Indonesia, economical value of sweet potato is low, compared with other starch sources. However, the sweet potatoes which are rich in starch made it a potential source of resistant starch. At present, various studies about resistant starch had been conducted. Recent studies showed that resistant starch consumption may reduce colorectal cancer risk.

Resistant starch has been defined as total amount of starch, and the products of starch degradation that resists digestion in the small intestine of healthy people. There are four types of resistant starch. Type 1 resistant starch (RS1) is starch that escapes digestion in the small intestine due to physical protection by the food matrix, such as grains. Type 2 resistant starch (RS2) is raw starch granules (ungelatinized) with compact structure which limits accessibility of digestive enzymes. Type 3 resistant starch (RS3) is retrogaded starch in which parts of the starch chains can crystallize into components that are less digestible. Most often, this occurs by cooking and cooling starch-containing foods. And the last, type 4 resistant starch (RS4) is not found naturally in foods. Starch that has been chemically modified to introduce bonds that are not digestible by human enzymes.

Starches that resist small intestinal breakdown are fermented by the resident bacteria in the large intestine, producing a variety of end products, the most significant of which is the short chain fatty acids (SCFA). SCFA, especially butyrate maintenance of colonic health and barrier function, butyrate has drawn most attention as this fatty acid is the major energy source for the colonocytes (Roediger 1990). Furthermore, butyrate has been shown to have anti-carcinogenic effects mainly by affecting proliferation, differentiation and apoptosis of colonocytes (Scheppach, et. al 1995).

There are several bacterial species which produce butyric acid. It is an anaerobic process. The genera Clostridium, Butyribacterium, and Butyrivibrio are the mostly studied microorganism. For commercial purposes, Clostridium species are preferred for butyric acid production. C. butyricum, C. tyobutyricum, C. thermobutyricum, C. beijerinckii, and C. populeti are of interest for the production of butyrate. Purwani, et. al (2012) observed that RS3 derived from sago and rice starch could be well utilized as substrate by Clostridium butyricum, and revealed that good proportion of acetate: propionat: butyrate in that fermentation.

Apoptosis occurs normally during development and aging and as a homeostatic mechanism to maintain cell populations in tissues and the cell is an active participant in its own demise (“cellular suicide”). Apoptosis is a form of programmed cell death characterized by cytoplasmic condensation, plasma membrane blebbing and nuclear pycnosis, leading to nuclear DNA breakdown into multiples of ~200 bp oligonucleosomal size fragments (Chen and Ioannou 1996).

One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result


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2

in the appearance of “DNA laddering” when the DNA is analyzed by agarose gel electrophoresis (Arends, et. al 1990). Principle of this assay is apoptotic DNA binds quickly to glass fiber fleece in the presence of a chaotropic salt, guanidine hydrochloride (guanidine HCl). After cellular impurities are washed off the fleece, the DNA is released from the fleece with a low salt buffer (Wyllie, et. al 1998).

B.

OBJECTIVE

The objective of this research was to analyze apoptotic DNA fragments of HCT-116 human colon cancer cell incubated with cell free supernatant contained short chain fatty acids (SCFA) derived from fermentation of type 3 resistant starch (RS3) of sweet potato (Ipomoea batatas) by C. Butyricum BCC B2571 using DNA ladder assay.


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3

II.

LITERATURE REVIEW

A.

RESISTANT STARCH (RS)

Resistant starch (RS) has been defined as total amount of starch, and the products of starch degradation that resists digestion in the small intestine of healthy people then enter the large bowel. It occurs for various reasons including chemical structure, cooking of food, chemical modification, and food mastication (Topping and Clifton 2001).There are four types of resistant starch. Starch found in plant cell walls that is inaccessible to amylase activity is one type of resistant starch, designated type 1 resistant starch (RS1). Food sources of RS1 include partially milled grains and seeds. Resistant starch also may be formed during food processing. Ungelatinized granules of starch are typically resistant to enzymatic digestion and are designated type 2 resistant starch (RS2). This type of starch can be found in potatoes and unripe (green) bananas. Cooking and cooling starchy foods by moist heat or extrude starchy foods, for example, generates retrogade starch called type 3 resistant starch (RS3). Chemical modifications of starch, such as formation of starch esters, or cross-bonded starches, also result in resistant starch, called type 4 resistant starch (RS4). RS3 and RS4 may be partially fermented by colonic bacteria (Gropper, et. al 2009). Interestingly, if RS2 granules are heated to over 100ºC, the granularity is lost and starch is gelatinized or the granules swell thereby increasing the availability of the starch to amylase. However, when the starch cools, there is some recrystallization of the starch. This is called retrogradation, which is resistant to α–amylase hydrolysis (Medeiros and Wildman 2012).

Starch that is trapped within whole plant cells or within the food matrix, and some starch granules that have not been fully gelatinized, are hydrolysed only very slowly by a-amylase and therefore may escape complete digestion in the small intestine. The energy yield to the body from this source is less than that provided if starch is digested and absorbed in the small intestine (Englyst, et. al 1996). The glycemic effects of starch is therefore lowered, which decreases insulin secretion. In addition to glycemic management resistant starches may play role in weight management as well (Medeiros and Wildman 2012).

The material included in the definition of RS reaches the human large intestine and thus becomes a substrate for microbial fermentation. The end-products are H2and CO2, CH4 in about half the population, and short-chain fatty acids. SCFA consist primarily of butyrate, propionate and acetate (Englyst, et. al 1996). SCFA contribute to normal large bowel function and prevent pathology through their actions in the lumen and on the colonic musculature and vasculature, through their metabolism by colonocytes (Topping and Clifton 2001).

B.

SHORT CHAIN FATTY ACID (SCFA)

Short chain fatty acids (SCFA) are end-product of luminal microbial fermentation of predominantly non-digestible dietary carbohydrates. SCFA with different carbon chain lengths (acetate (C2), propionate (C3), butyrate (C4), valerate (C5), and caproate (C6)) are produced in


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4

varying amounts depending on the diet and the composition of the intestinal microbiota (Marleen,

et. al 2003). SCFA constitute approximately two-thirds of the colonic anion concentration (70-130 mmol/l), mainly as acetate, propionate, and butyrate (Mortensen and Clausen 1996). Various population data show that SCFA production is in order of acetate > propionate > butyrate in a molar ratio of approximately 60:20:20 or 3:1:1, respectively in the proximal and distal colon (Cummings, et. al 1987; Topping and Clifton 2001).

The major source of this in the human colon is thought to be plant cellwall polysaccharides such as cellulose, pectins, and hemicelluloses, currently referred to in human nutrition as dietary fibre. Starch would also be a suitable substrate if it were to reach the colon in significant quantities. Plant cell-wall polysaccharides are composed of hexose (glucose and galactose), pentose (xylose and arabinose), and uronic acid monomers which are fermented by gut micro-organisms along a variety of pathways. The important feature to remember of this metabolism is that it is anaerobic. Many lines of evidence therefore support the idea that fermentation occurs in the colon but, because of the difficulties in obtaining access to the human large bowel during normal digestion, most of the evidence for fermentation has, of necessity, been indirect (Cummings, et. al 1987).

There are several bacterial species which produce butyric acid. Most butyric acid-producing bacteria form acetic acid in addition to butyric acid as their major fermentation products.

Clostridium butyricum, C. tyobutyricum, C. thermobutyricum, C. beijerinckii, and C. populeti are of interest for the production of butyrate. There are some benefits using Clostridium butyricum.

These bacteria needs simple growth medium, produces high metabolite, and easy to isolate. Therefore, Clostrium butyricum also has butyrogenic activity, produce more butyric acid than acetic acid and propionic acid. C. butyricum had the highest specific growth rates compared with other genera. On the other hand, Clostridium spp. are generally less dominant in the human colon and hence the role of Clostridium in the production of butyrate in humans is unclear (Wang 1999).

SCFA will be absorbed and contribute about 3 kilocalories per gram (Medeiros and Wildman 2012). SCFA are rapidly absorbed and have shown to have distinct bioactivity depending on their chain length. With regard to maintenance of colonic health and barrier function, butyrate has drawn most attention as this fatty acid is the major energy source for the colonocytes (Roedriger 1990). Furthermore, butyrate has been shown to have anti-carcinogenic effects mainly by affecting proliferation, differentiation, and gene expression of colonocytes (Scheppach, et. al 1995). Butyrate concentration didn‟t show a significant correlation with pH or

propionic concentration (McIntyre, et. al 1993). The production of propionate is by two main, but circuitous, routes, firstly, involving fixation of CO2, to form succinate which is subsecquently decarboxylated (the 'decarboxylic acid pathway') or secondlly, from lactate and acrylate (the 'acrylate pathway'). Acetate is usually formed by the oxidative decarboxylation of pyruvate and butyrate by reluction of acetoacetate formed from acetate (Cummings, et. al 1981).

Short chain fatty acids are metabolized at 3 major sites in the body (Hijova and Chmelarova 2007):


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1. Cells of the ceco-colonic epithelium that use butyrate as a major substrate for the maintenance of energy producing path-ways;

2. Liver cells that metabolize residual butyrate with propionate used for gluconeogenesis; 50% to 70% of acetate is also taken up by the liver;

3. Muscle cells that generate energy from the oxidation of residual acetate.

In ruminants and other herbivores, SCFA are absorbed and transported via the portal vein to the liver, and the fraction not absorbed is distributed to the other body organs and tissues for metabolism. In herbivores, peripheral venous SCFA concentrations are high due to comparatively low visceral extraction and high rates of absorption into the circulation. However, human peripheral venous blood concentrations are normally low, and only acetate is present in measurable amounts. This profile reflects the lower SCFA production rates and greater visceral extraction in omnivores, meaning that human peripheral venous SCFA are not representative of those in the portal circulation. Human experimentation has been confined largely to fecal measurements, which are also limited as 95% of SCFA are produced and absorbed within the colon (Topping and Clifton 2001).

Purwani, et. al (2012) study revealed that type 3 resistant starch (RS3) derived from sago and rice starch that treated with pullulanase could be well utilized as substrate by Clostridium butyricum and showed good proportion of acetate: propionate: butyrate in the bacterial culture filtrate. The fermentation of resistant starch type 3 (RS3) derived from sago starch treated with pullulanase (RSSP) by C. butyricum produced 17.65 mM of butyrate, and fermentation of RS3 derived from rice starch treated with pullulanase (RSRP) by C. butyricum produced 21.76 mM, higher concentration than fermentation of RSSP. This study implied that difference of starch source resulted difference of SCFA concentration.

C.

APOPTOSIS

Apoptosis (“normal” or “programmed” cell death) is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Apoptosis is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise (“cellular suicide”) (Wyllie, et. al 1998). Apoptosis is a form of programmed cell death characterized by cytoplasmic condensation, plasma membrane blebbing and nuclear pycnosis, leading to nuclear DNA breakdown into multiples of ~200 bp oligonucleosomal size fragments (Chen and Ioannou 1996).


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6

Figure 1 Illustration of the morphological features of apoptosis (Wyllie, et. al 1998)

Apoptosis is characterised by an initial shrinkage of the cell, which in a tissue means breaking cell-to-cell contacts with neighbours and rounding up. The consequence of this stage tends to be that the volume of the cell becomes smaller, and the cytoplasmic internal membranes, ribosomes, mitochondria, and other cytoplasmic organelles are more concentrated in the cytoplasm, which then consequently looks darker. The organelles remain intact and healthy looking very late into the process of death, suggesting that the cell continues metabolic activity for some considerable time. This indeed can be demonstrated in many cell systems by using various inhibitors of metabolic processes (RNA and protein synthesis inhibitors), which delay the progression of a cell through the apoptotic sequence. Apoptosis characteristically involves single isolated cells and not clusters. The DNA or chromatin material in the nucleus condenses very extreme (Potten and Wilson 2004). Pyknosis is the result of chromatin condensation and this is the most characteristic feature of apoptosis. On histologic examination with hematoxylin and eosin stain, apoptosis involves single cells or small clusters of cells. The apoptotic cell appears as a round or oval mass with dark eosinophilic cytoplasm and dense purple nuclear chromatin fragments. Electron microscopy can better define the subcellular changes. Early during the chromatin condensation phase, the electron-dense nuclear material characteristically aggregates peripherally under the nuclear membrane although there can also be uniformly dense nuclei.

Extensive plasma membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies during a process called “budding.” Apoptotic bodies consist of cytoplasm with tightly packed organelles with or without a nuclear fragment. The organelle integrity is still maintained and all of this is enclosed within an intact plasma membrane. These bodies are subsequently phagocytosed by macrophages, parenchymal cells, or neoplastic cells and degraded within phagolysosomes. Macrophages that engulf and digest apoptotic cells are called “tingible body macrophages” and are frequently found within the reactive germinal centers of lymphoid follicles or occasionally within the thymic cortex. The tingible bodies are the bits of nuclear debris from the apoptotic cells. There is essentially no inflammatory reaction associated with the process of apoptosis nor with the removal of apoptotic cells because: (1) apoptotic cells


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7

do not release their cellular constituents into the surrounding interstitial tissue; (2) they are quickly phagocytosed by surrounding cells thus likely preventing secondary necrosis; and (3) the engulfing cells do not produce anti-inflammatory cytokines (Savill and Fadok 2000; Kurosaka, et. al 2003).

The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, an irreversible event that commits the cell to die and occurs before changes in plasma membrane permeability (prelytic DNA fragmentation). The fragmentation of chromosomal DNA is a hallmark of apoptosis and may facilitate apoptosis by terminating DNA replication and gene transcription. In many systems, this DNA fragmentation has been shown to result from activation of an endogenous Ca2+ and Mg2+ dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites located between nucleosomal units (linker DNA) generating mono- and oligonucleosomal DNA fragments (Arends et. al, 1990; Wyllie, et. al 1998).

In priciple, there are two main apoptotic pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. In both pathways, cysteine aspartyl-specific proteases (caspases) are activated that cleave cellular substrates, and leads to the biochemical and morphological changes that are characteristic of apoptosis. However, there is now evidence that the two pathways are linked and that molecules in one pathway can influence the other (Igney and Krammer 2002). Figure 2 shows the two main apoptotic signalling pathways.


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8

Apoptotic cells exhibit several biochemical modifications such as protein cleavage, protein cross-linking, DNA breakdown, and phagocytic recognition that together result in the distinctive structural pathology described previously (Hengartner 2000). Caspases are widely expressed in an inactive proenzyme form in most cells and once activated can often activate other procaspases, allowing initiation of a protease cascade. Some procaspases can also aggregate and autoactivate. This proteolytic cascade, in which one caspase can activate other caspases, amplifies the apoptotic signaling pathway and thus leads to rapid cell death (Elmore 2007).

Although DNA fragmentation into oligonucleosomal ladders is characteristic of apoptosis, recent evidence indicates that not all cells undergo such extensive DNA fragmentation (Cohen, et. al1. 1992 in Chen and Ioannou 1996). In fact, fragmentation of DNA into kilobase-size fragments appears to be an early event in apoptosis, preceding the complete digestion of DNA into multiples of nucleosomal size fragments (Cohen and Sun 1994 in Chen and Ioannou 1996).

D.

DNA LADDER ASSAY

One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of “DNA laddering” when the DNA is analyzed by agarose gel electrophoresis (Arends, Morris, and Wyllie 1990). Principle of this assay is apoptotic DNA binds quickly to glass fiber fleece in the presence of a chaotropic salt, guanidine hydrochloride (guanidine HCl). After cellular impurities are washed off the fleece, the DNA is released from the fleece with a low salt buffer (Wyllie, et. al 1998).

DNA ladder is asolution of DNA molecules of different lengths used in agarose gel electrophoresis. it is applied to an agarose gel as a reference to estimate the size of unknown DNA molecules. In addition it can be used to approximate the mass off a band by comparison to a special mass ladder. Different DNA ladders are commercially available depending on expected DNA length. The 1 kb ladder with fragment ranging from about 0.5 kbp to 10 or 12 kbp and the 100 bp ladder with fragment ranging from 100 bp to just above 1000 bp are the most frequent. DNA ladders are often produced by a suitable restriction digest of plasmid.

DNA electrophoresis is an analytical technique used to separate DNA fragments by size. An electric field forces the fragments to migrate through a gel. DNA molecules normally migrate from negative to positive potential due to the net negative potential due to the net negative charge of the phosphate backbone of the DNA chain. At the scale of the length of DNA molecules, the gel looks much like a random, intricate network. Longer molecules migrate more slowly because they are more easily „trapped‟ in the network. After the separation is completed, the fractions of DNA fragments of different length are often visualized using a fluorescent dye spesific for DNA, such as ethidium bromide. The gel shows bands corresponding to different DNA molecules populations with different molecular weight. Fragment size is usually reported in “nucleotides”, “base pairs” or


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separated. Fragment size determination is typically done by comparison to commercially available DNA ladders containing linear DNA fragments of known length.

The types of gel most commonly used for DNA electrophoresis are agarose (for relatively long DNA molecules) and polyacrylamide (for high resolution of short DNA molecules, for example in DNA sequencing). Electrophoresis techniques used in the assessment of DNA damage include alkaline gel electrophoresis and pulsed field gel elctrophoresis. The measurement and analysis are mostly done with a specialized gel analysis software.

DNA molecules must be separated from other cellular material before they can be examined. Cellular proteins that package and protect DNA in the environment of the cell can inhibit the ability to analyze the DNA. Therefore, DNA extraction methods have been developed to separate proteins and other cellular materials from the DNA molecules. The quantity and quality of DNA often need to be measured prior to proceeding further with analytical procedures to ensure optimal results. Organic extraction was for many years the most widely used methods for DNA extraction. Organic extraction involves the serial addition of several chemicals. First sodium dodecylsulfate (SDS) and proteinase K are added to break open the cell walls and to break down the proteins that protect the DNA molecules while they are in chromosomes (Butler 2010). NA sample purity and molecular weight must be controlled by agarose gel electrophoresis with 0.4-0.8% ethidium bromide stain. The DNA can be suspended in TE buffer (Tris-HCL and EDTA pH 8.0) or double-distilled water (Walker and Rapley 2008).


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Herrmann, et. al (1994) compared the methods for isolation of apoptotic DNA fragments. The research showed that method for isolation of apoptotic DNA affected the bands of DNA fragmentation. Figure 5 shows that the NP-40 lysis method with a frequently used method for isolation of apoptotic. It shows that when NP-40 lysis method produces better DNA fragmentation (figure 5 lane 4-7) compares with phenol extraction procedures (figure 5 lane 1-3). This research used ethanol extraction procedure. The fragmentation of DNA in this research was similar not clear. This research still needs optimazion methodology of DNA extraction to get the best result of DNA fragmentation assay.


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V.

CONCLUSION AND RECOMMENDATION

A.

CONCLUSION

Based on this research, Short Chain Fatty Acids (SCFA) derived from fermentation of type-3 resistant starch of Ipomoea batatas containing 1,00 and 1, 25 mM butyrate produced DNA bands different from the control when measured with DNA ladder assay. The DNA bands of the treated cells appeared as longer smear. There were separate DNA bands in the floating cells in the treated medium (SCFA containing 0.625, 1.00, and 1.25 mM butyrate) at ±100 bp. These bands were not presence in the control. DNA fragmentation was not clear in the attached cells, but was observed as a separate bands in the floating cells in the medium. This imply that SCFA might induce apoptosis at very early stage in the attached cells and later stage in the floating cells in the medium.

B.

RECOMMENDATION

DNA fragmentation in this research was not clearly/optimally observed. This might relate to suboptimal DNA extraction. Isolation of apoptotic cells and optimazion methodology of DNA extraction should be done to get the best result of DNA fragmentation assay.


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REFERENCES

Arends MJ, Morris RG, and Wyllie AH. 1990. Apoptosis: the role endonuclease. American Journal Pathology. 136: 593-608.

Boffa LC, Vidali G, Mann RS and Allfrey VG. 1978. Suppression of histone deacetylation in vivo and in vitro by sodium butyrate. The Journal of Biological. Chemistry. 253 (10): 3364-3366. Butler JM. 2010. Fundamentals of Forensic DNA Typing. Elsevier Inc, United States of America. Chen and Ioannou. 1996. Quantitation of DNA fragmentation in apoptosis. Nucleic Acids

Research. 24(5): 992-993.

Cummings JH. 1981. Short chain fatty acids in the human colon. Gut. 22: 763-769.

Cummings JH, Pomare EW, Branch WJ, Naylor CPE, and Macfarlane GT. 1987. Short chain fatty acids in human large intestine, portal, hepatic, and venous blood. Gut. 28: 1221-1227. Daly K and Shirazi-Beechey SP. 2006. Microarray analysis of butyrate regulated genes in colonic

epithelial cells. DNA and Cell Biol. 25: 49-62.

Elmore Susan. 2007. Apoptosis: A review of programmed cell death. Toxicol Pathol. 35(4): 495– 516.

Englyst H N, Kingman S M, Hudson G J, and Cummings J H. 1996. Measurement of resistant starch in vitro and in vivo. British Journal of Nutrition. 75: 749-755.

Gropper SS, Smith JL, and Groff JL. 2009. Advanced Nutrition and Human: Fifth Edition. Cengage Learning, Canada.

Hengartner MO. 2000. The biochemistry of apoptosis. Nature. 407: 770-776.

Herrmann M, Lorenz H M, Voll R, Grunke M, Woith W, and Kalden J R. 1994. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Research. 22 (24): 5506-5507.

Hijova E and Chmelarova A. 2007. Short chain fatty acids and colonic health. Bratisl Lek Listy. 108 (8): 354-358.

Hinnebusch BF, Meng S, Wu JT, Archer SY, Hodin RA. 2002. The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation. American Society for Nutritional Science. 132:1012-1017.

Kurosaka Kahori, Takahashi Munehisa, Watanabe Naoko, and Kobayashi Yoshiro. Silent cleanup of very early apoptotic cells by macrophages. The Journal of Immunology. 171: 4672-4679. Marleen HMC, Nuenen van, Meyer PD, and Venema Koen. 2003. The effect of various inulins

and Clostridium difficile on the metabolic activity of human colonic microbiota in vitro. Microbial Ecology in Health an Disease. 15: 137-144.

McIntyre A, Gibson PR, and Young GP. 1993. Butyrate production from dietary fibre and protection againts large bowel cancer in a rat model. Gut. 34: 386-391.

Medeiros DM and Wildman REC. 2012. Human Nutrition: Second Edition. Jones and Bartlett Learning LLC, United States of America.


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Mortensen PB and Clausen MR. 1996. Short-chain fatty acids in the human colon: relation to gastrointestinal health and disease. Scandinavian Journal of Gastroenterology. 216: 132-148. Potten Christoper and Wilson James. 2004. Apoptosis: The Life and Death of Cells. Cambridge

University Press, New York.

Purwani EY and Suhartono MT. 2010. Study on resistant starch type 3 derived from rice and sago, as functional food ingredient with capability to prevent colorectal ancer (CRC) disease. Final report doctorate research grant (unpub).

Purwani EY, Purwadaria T, Suhartono MT. 2012. Fermentation RS3 derived from sago and rice starch with Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629. Anaerobe 18(1):55-61.

Roediger WEW. 1990. The starved colon--diminished mucosal nutrition, diminished absorption, and colitis. Dis Colon Rectum. 33: 858-862.

Ruemmele FM, Dionne S, Qureshi I, Sarma DSR, Levy E, Seidman EG. 1999. Butyrate mediates Caco-2 cell apoptosis via up-regulation of pro-apoptotic BAK and inducing caspase-3 mediated cleavage of poly-(ADP-ribose) polymerase (PARP). Cell Death and Differentiation 6:729-735.

Savil John and Fadok Valerie. 2000. Corpse clearance defines the meaning of cell death. Nature. 407: 784-788.

Scheppach W, Bartram HP, Richter F. 1995. Role of short-chain fatty acids in the prevention of colorectal cancer. Eur J Cancer. 31A: 1077-1080.

Topping D L and Clifton P M. 2001. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiological Reviews. 81(3): 1031-1064. Van Nuenen MHC, Meyer D, Venema K. The effect of various inulins and clostridium dificile on

the metabolic activity of the human colonic microbiota in vitro. Microbial Ecology in Health and Disease 2003;15:137-44.

Walker JM and Rapley Ralph. 2008. Molecular Biomethods Handbook: Second Edition. Human Press, United States of America.

Wang Xin, Conway PL, Brown IL, and Evans AJ. 1999. In vitro utilization of amylopectin and high-amylose maize (amylomaize) starch granules by human colonic bacteria. App and Environmental Microbiology. 65 (11): 4848-4854.

Wyllie Andrew, Donahue Vicki, Fischer Betram, Hill David, Keesey Joe, and Manzow Simone. 1998. Apoptosis and Cell Proliferation: 2nd Edition. Mannheim Boehringer, Germany.


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REFERENCES

Arends MJ, Morris RG, and Wyllie AH. 1990. Apoptosis: the role endonuclease. American Journal Pathology. 136: 593-608.

Boffa LC, Vidali G, Mann RS and Allfrey VG. 1978. Suppression of histone deacetylation in vivo and in vitro by sodium butyrate. The Journal of Biological. Chemistry. 253 (10): 3364-3366. Butler JM. 2010. Fundamentals of Forensic DNA Typing. Elsevier Inc, United States of America. Chen and Ioannou. 1996. Quantitation of DNA fragmentation in apoptosis. Nucleic Acids

Research. 24(5): 992-993.

Cummings JH. 1981. Short chain fatty acids in the human colon. Gut. 22: 763-769.

Cummings JH, Pomare EW, Branch WJ, Naylor CPE, and Macfarlane GT. 1987. Short chain fatty acids in human large intestine, portal, hepatic, and venous blood. Gut. 28: 1221-1227. Daly K and Shirazi-Beechey SP. 2006. Microarray analysis of butyrate regulated genes in colonic

epithelial cells. DNA and Cell Biol. 25: 49-62.

Elmore Susan. 2007. Apoptosis: A review of programmed cell death. Toxicol Pathol. 35(4): 495– 516.

Englyst H N, Kingman S M, Hudson G J, and Cummings J H. 1996. Measurement of resistant starch in vitro and in vivo. British Journal of Nutrition. 75: 749-755.

Gropper SS, Smith JL, and Groff JL. 2009. Advanced Nutrition and Human: Fifth Edition. Cengage Learning, Canada.

Hengartner MO. 2000. The biochemistry of apoptosis. Nature. 407: 770-776.

Herrmann M, Lorenz H M, Voll R, Grunke M, Woith W, and Kalden J R. 1994. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Research. 22 (24): 5506-5507.

Hijova E and Chmelarova A. 2007. Short chain fatty acids and colonic health. Bratisl Lek Listy. 108 (8): 354-358.

Hinnebusch BF, Meng S, Wu JT, Archer SY, Hodin RA. 2002. The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation. American Society for Nutritional Science. 132:1012-1017.

Kurosaka Kahori, Takahashi Munehisa, Watanabe Naoko, and Kobayashi Yoshiro. Silent cleanup of very early apoptotic cells by macrophages. The Journal of Immunology. 171: 4672-4679. Marleen HMC, Nuenen van, Meyer PD, and Venema Koen. 2003. The effect of various inulins

and Clostridium difficile on the metabolic activity of human colonic microbiota in vitro. Microbial Ecology in Health an Disease. 15: 137-144.

McIntyre A, Gibson PR, and Young GP. 1993. Butyrate production from dietary fibre and protection againts large bowel cancer in a rat model. Gut. 34: 386-391.

Medeiros DM and Wildman REC. 2012. Human Nutrition: Second Edition. Jones and Bartlett Learning LLC, United States of America.


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19

Mortensen PB and Clausen MR. 1996. Short-chain fatty acids in the human colon: relation to gastrointestinal health and disease. Scandinavian Journal of Gastroenterology. 216: 132-148. Potten Christoper and Wilson James. 2004. Apoptosis: The Life and Death of Cells. Cambridge

University Press, New York.

Purwani EY and Suhartono MT. 2010. Study on resistant starch type 3 derived from rice and sago, as functional food ingredient with capability to prevent colorectal ancer (CRC) disease. Final report doctorate research grant (unpub).

Purwani EY, Purwadaria T, Suhartono MT. 2012. Fermentation RS3 derived from sago and rice starch with Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629. Anaerobe 18(1):55-61.

Roediger WEW. 1990. The starved colon--diminished mucosal nutrition, diminished absorption, and colitis. Dis Colon Rectum. 33: 858-862.

Ruemmele FM, Dionne S, Qureshi I, Sarma DSR, Levy E, Seidman EG. 1999. Butyrate mediates Caco-2 cell apoptosis via up-regulation of pro-apoptotic BAK and inducing caspase-3 mediated cleavage of poly-(ADP-ribose) polymerase (PARP). Cell Death and Differentiation 6:729-735.

Savil John and Fadok Valerie. 2000. Corpse clearance defines the meaning of cell death. Nature. 407: 784-788.

Scheppach W, Bartram HP, Richter F. 1995. Role of short-chain fatty acids in the prevention of colorectal cancer. Eur J Cancer. 31A: 1077-1080.

Topping D L and Clifton P M. 2001. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiological Reviews. 81(3): 1031-1064. Van Nuenen MHC, Meyer D, Venema K. The effect of various inulins and clostridium dificile on

the metabolic activity of the human colonic microbiota in vitro. Microbial Ecology in Health and Disease 2003;15:137-44.

Walker JM and Rapley Ralph. 2008. Molecular Biomethods Handbook: Second Edition. Human Press, United States of America.

Wang Xin, Conway PL, Brown IL, and Evans AJ. 1999. In vitro utilization of amylopectin and high-amylose maize (amylomaize) starch granules by human colonic bacteria. App and Environmental Microbiology. 65 (11): 4848-4854.

Wyllie Andrew, Donahue Vicki, Fischer Betram, Hill David, Keesey Joe, and Manzow Simone. 1998. Apoptosis and Cell Proliferation: 2nd Edition. Mannheim Boehringer, Germany.