Fibersol-2 induces apoptosis of Apc-deficient colorectal Cancer (SW480) cells and decreases polyp formation in Apc MIN mice

ABSTRACT

The consumption of dietary fibers has been implicated with a lowered risk of human colorectal cancer. Proposed mechanisms involve alterations in the stool consistency, transit time, and formation of short-chain fatty acid by dietary fiber fermentation, and the reorganization of gut microbiota. Here we show that Fibersol-2, a digest-resistant maltodextrin, not only inhibits proliferation of colorectal SW480 cancer cell lines by increasing reactive oxygen species (ROS), but decreases the numbers of the adenoma count in Multiple Intestinal Neoplasia (MIN) mice carrying a mutation in the Adenomatous Polyposis Coli gene by 84 d of age. These observations provide direct evidence that Fibersol-2 intrinsically contains anti-cancer activity, independent of the intestinal metabolism and any potential interactions with the microbiota.

KEYWORDS:

• Apc
• apoptosis
• cancer
• fibersol
• maltodextrin
• MIN

Introduction

Human colorectal cancer (CRC) is the second cause of cancer mortality in the USA.¹ The incidence of CRC is typically associated with the accumulation of mutations in oncogenes and tumor suppressor genes.^2,3 In CRC specifically, one of the commonly affected tumor suppressor genes is Adenomatous Polyposis Coli (Apc) occurring during the early stages of CRC.⁴ Apc plays an essential role in the Wnt/β-catenin signaling pathway ^5,6 which regulates the stabilization and accumulation of β-CATENIN protein in the nucleus and the cytoplasm. Nuclear β-CATENIN is the hallmark of an active canonical Wnt pathway. The majority of all cases of CRC have a mutation of the canonical Wnt pathway. Without the presence of Wnt signaling, cells regulate β-Catenin activity via a multiprotein complex, which can phosphorylate β-CATENIN, targeting it for ubiquitination and subsequent degradation.⁷ The β-CATENIN complex is formed by APC, AXIN and GSK3β. AXIN and APC form a structural scaffold that allows GSK3β to phosphorylate β-CATENIN, as well as APC and AXIN. When Wnt ligand binds to its receptor, FRIZZLED (Fz), it triggers a signaling cascade destabilizing the degradation complex now allowing unphosphorylated β-CATENIN to accumulate and translocate into the nucleus, where it acts as a cofactor for T-cell-/lymphoid enhancing factor (TCF/LEF).⁸ This triggers transcriptional activation of various genes related to cell fate and cell proliferation.^5,10-13 80-90% of patients with sporadic CRC do show a mutation in the Apc gene.^14,15 APC is a large protein (312 kDa) that has multiple and diverse functions in cell cycle regulation, chromosome stability, cell migrations and adhesion.^9,11-13

In the current study we investigated the effects of Fibersol-2 (FS2) on cells with mutated Apc in cell culture and the pathophysiology in Apc mutant mice.¹⁶ Dietary fiber supplements have long been associated with improved intestinal function and reduced risk for CRC. Fibersol-2 (FS2), a digestion resistant maltodextrin, is a soluble corn fiber which is not digested or absorbed in the human small intestine, passing to the large intestine, it is fermented producing short-chain fatty acids, lower pH levels, gaseous byproducts and effecting the microbiota. ¹⁷ Clinical studies have shown that it helps by decreasing the postprandial blood glucose spikes and insulin levels and retaining healthy serum triglycerides levels.¹⁸

Here, an Apc mutant colon cancer cell line (SW480)^19,20 and C57BL/6J mice spontaneously developing multiple intestinal neoplasia (MIN) caused by an ENU induced mutation in the Apc gene ^21,22 were used for analyzing the effects of FS2 in cell culture and in vivo. We found that FS2 treatment increases apoptosis and ROS of SW480 cells. In vivo MIN mice studies demonstrated that FS2 treatment significantly decreases the formation of adenomas in the small intestine and reduces the number of polyps in the colon of the animals.

Material and methods

Cells and reagents

SW480 (human colorectal adenocarcinoma) cells were purchased from ATCC (CCL-228). The cells were grown in Leibovitz's L-15 Medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin (100 units/ml, Invitrogen) and 10 mM sodium pyruvate (Sigma, St. Louis, MO).

Fibersol-2 was provided by Matsutani America (Itasca, IL), and solubilized in autoclave sterile water for mouse feeding.

ROS detection and apoptosis

SW480 cells were treated with or without Fibersol-2 (5% final concentration). After 24 hours culture ROS and apoptosis were examined.

MitoSOX (mitochondrial superoxide indicator), and CM-H2DCFDA (general oxidative stress indicator) were obtained from Invitrogen. For measurement of mitochondrial and cellular ROS cells were cultured in complete DMEM containing 5 μM Mitosox or CM-H2DCFDA for 10 min at 37°C protected from light. Cells were detached by 0.1 trypsin/EDTA solution after incubation and cell suspensions were analyzed by flow cytometry.

Apoptosis was determined by annexinV/ Propidium Iodide (PI) double staining kit (BD Biosciences) according to manufacturer's instruction by a flow cytometer (LSR II, BD Biosciences, Franklin Lakes, NK). The data were analyzed with FCS Express 4 program (De Novo software Los Angeles, CA).

Apc mutant mice

Breeder pairs of C57BL/6J Apc^MIN/+ mice were obtained from Dr. William Dove (UW-Madison, Madison, WI).²¹ The mice used were bred and maintained at University of Wisconsin Parkside (Kenosha, WI) under a constant LD (light/dark) 12hours/12hours cycle and the experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC). New litters were weaned at 21 d of age, separated by sex and grouped housed no more than 4 per cage under the same light/dark cycle. Males were housed in a separate room from females. Mice were fed a diet containing 10% fat (Lab Diet 5 K20) recommended by Dr. Dove to support the MIN phenotype.

Water bottles were filled with either regular tap water (control group) or Fibersol-2 (1% final concentration) (treated group). All efforts were made to achieve a 50/50 ratio of treated vs. control offspring produced from the same male MIN breeder. At weaning, Fibersol-2 treatment was initiated by supplementing the drinking water. The 1% concentration of Fiberol-2 was established as optimal for water supplementation in a feasibility study (data not shown) and is lower than the optimal dosage used in the cell culture studies.

All mice were euthanized at 84 d of age using a flow of CO₂ gas to effect. The intestinal tract, cecum and colon were removed. Cecums were stored at −20°C. Intestinal tract and colon were placed in a petri dish containing 1X PBS solution. The intestinal tract was divided in 4 equal parts and placed on bibulous paper saturated with PBS. Tracts and colons were split open longitudinally with micro-scissors and spread open. Debris within was washed out with a flow of PBS interchanged with 70% ethanol (a pre-fixation of tissue). Papers were stacked, rolled up and stored in a 50 ml conical tube containing 10% formalin for at least 24 hours. Formalin solution was replaced with 70% ethanol for indefinite storage.

Blind scoring of the intestinal adenomas occurred any time after this point. One animal's tissue was removed from the conical tube; the tissue was removed from the paper and placed in a Petri dish containing 70% ethanol. The tissue was viewed using a dissecting microscope under a 10X magnification. The number of intestinal tumors in each segment of the small intestine and the colon were determined and recorded. The tissue was returned to the conical tube and stored.

Results

Fibersol-2 increases ROS generation in SW480

ROS are byproducts of normal cell metabolism. Mitochondria generate ATP by oxidizing lipids, amino acids and glucose, and also generate O₂ by transferring electrons to the electron transport chain (ETC). But, in this process, free radicals are also generated. Excessive ROS generation, or failure of oxidant scavenging systems, can disrupt cellular function by causing oxidation of proteins, DNA and lipids. Lower levels of oxidants act as signal transduction messengers in redox signaling pathways, which have important roles in the regulation of cell function, including proliferation. ^23,24 Cancer-inducing mutations in mitochondrial enzymes can augment the generation of ROS from multiple sites. Several studies suggest a direct relationship between ROS and cancer development; free radicals can induce DNA mutation resulting in genomic instability and carcinogenesis.²⁵ Cancer cells have higher levels of ROS resulting from their increased metabolic activity, peroxisome activity, mitochondrial dysfunction, oncogene activity, increased activity of oxidases, etc.^26-28 Previous studies of our group have demonstrated that FS2 can reduce the mitochondrial membrane potential causing mitochondrial malfunction and increased mitochondrial ROS production ²⁹ in HCT116 and HCT116 p53(-) cell lines.³⁰ In the current study the effects of FS2 on the SW480 cell line was investigated. SW480 cells carry mutations in both the p53 and the Apc genes.³¹ Basal levels of cellular and mitochondrial ROS were determined by CM-H₂DCFDA and MitoSox respectively. CM-H₂DCFDA has been used extensively to measure cellular H₂O₂ and other ROS, but it does not measure superoxide directly,³² on the other hand, MitoSOX red is a selective indicator of mitochondrially-generated superoxide production.³⁰ The SW480 cells were either treated with FS2 (final concentration 5%) or with water (control) for 24 hours. An increase of 15% in the mitochondria ROS production (using MitoSox) after FS2 treatment was observed in FS2 treated SW480 cells. In order to determine if the cause of the observed increase in ROS was due to mitochondria or other organelles responding to FS2 treatment, CM-H₂DCFDA ROS production was determined to measure the total cellular ROS formation. An increase in total ROS was observed after FS2 treatment and that increase is 10% higher that the observed superoxide production (25% more than untreated cells). These results strongly suggest that ROS is generated not only in mitochondria, and that some other organelles are producing ROS when treated with FS2 (Fig. 1).

Figure 1. Shown are the levels of ROS using MitoSOX (mitochondrial superoxide indicator) (A) and CM-H2DCFDA (general oxidative stress indicator) (B) without and with 5% FS2 treatment. FS2 leads to an increase in the amount of ROS..

Fibersol-2 increases apoptosis in SW480 cells

It has been well demonstrated that many of the therapeutic reagents increase cellular ROS levels, causing DNA damage and subsequent apoptosis of cancer cells. Previous results of our group have shown that FS2 induce activation of caspase 3 and 9 in HCT116 human colorectal cancer cells and this activation is p53/Bax dependent.²⁹ In the present study we looked at whether the increased levels of ROS induced by FS2 treatment subsequently can cause apoptosis of SW480 colorectal cancer cells. Cells were treated with FS2 (final concentration 5%) or water (as a control) for 48 h. Apoptosis activity was analyzed by Annexin V/PtdIns double staining and flow cytometry detection. As shown in Fig. 2 apoptosis is 3.75 fold higher in SW480 treated with FS2 than in untreated cells.

Figure 2. Shown is the measured apoptosis rates analyzed by Annexin V/PI double staining and flow cytometry detection (A+B). Treatment with FS2 significantly increased the apoptosis rate inSW480 cells treated by 3.75 fold compared to water treated controls (C).

Fibersol-2 decreases the number of adenomas in the small intestine of Apc^MIN/+ mutant mice

In order to examine if FS2 exhibits inhibitory characteristics in vivo, drinking water supplemented with 1% FS2 was provided to mice carrying a heterozygous mutant set of the Apc gene (Apc^MIN/+ ), which typically show high levels of adenomas in their digestive tract within 84 d of age.²¹ Apc^MIN/+ mice present pathological differences in the small-intestinal and colonic regions ^33,34 as well as female and male specific variances.^35-37 Thus, FS2s anti-tumor activity was studied in both male and female Apc^MIN/+ mice. Three week old mice were given 1% final concentration of FS2 in the drinking water for a total duration of 9 weeks before euthanasia. The intestinal tract from duodenum to colon was harvested. The duodenum, jejunum and ileum were analyzed under the dissecting microscope and the total small intestinal adenoma count for each animal was determined. An example of an adenoma in the small intestinal region is shown in Fig. 3. The group averages (control vs FS2, males vs females) are presented in Fig. 4. Female Apc^MIN/+ mice typically show a higher intestinal tumor count compared to their male littermates.^35,37 In the present study a 2 factor ANOVA confirmed a significant prevalence for intestinal tumors in female mice (gender; F = 5.844, p = 0.017). At the same time, animals treated with 1% FS2 showed a significant reduction in total adenoma counts (FS2; F =5.985, p=0.015) compared to the water treated groups, but we found no interaction between gender and treatment (F=0.029, p=0.865) (see Table 1).

Figure 3. 10x magnification of histological samples of a small intestine and colon from an 84 d old Apc^MIN/+ mouse. The small intestinal tract was divided into 4 equal parts and placed on bibulous paper saturated with PBS. Tracts and colons were split open longitudinally with micro-scissors and spread open. Debris within was washed out with a flow of PBS interchanged with 70% ethanol (a pre-fixation of tissue). Samples were scored blind and are reported on Table 1. (A) The arrow point toward 2 examples of adenomas. (B) Two polyps can be clearly identified as dark spots on the tissue (arrows). Polyps were more numerous in male mice and are absent in C57BL/6J wildtype animals.

Figure 4. Total average counts in the small intestine for each group are shown. The error bars indicate the Standard Error from the Mean (SEM). The dotted and the solid lines indicated male and female animals respectively. Females displayed a significantly higher number of tumors over males, and a significant reduction of counts was observed between the control (H₂O) and the 1%FS2 treated animals.

Table 1. The 4 tested groups; males, females with and without the administration of 1% fibersol 2 are shown in this table. The n for each group and the average total tumor counts and the Standard deviations from the mean are indicated.

			average number of counts
Treatment	Gender	Number of mice(n)	in small instestine	SD	in colon	SD
water	male	43	81	26.2	3.9	1.9
1% fibersol 2 (FS2)	male	45	71	28.6	3	1.7
water	female	40	93	32.7	2.5	1.7
1% fibersol 2 (FS2)	female	46	80	32	2.3	1.8

Fibersol-2 decreases the number of polyps in the colon in male and female Apc^MIN/+ mutant mice

In the colon the Apc mutation leads to an increased development of polyps. In humans, mutations of Apc have been associated with familial adenomatous polyposis (FAP) or Gardner's syndrome.^38,39 While the development of adenomas in the small intestine in affected mice is rapid, we investigated if the development of colonic adenomatous polyposis is also affected by FS2. We compared Apc^MIN/+ colon tissues of FS2 treated versus untreated mice independently and the results are summarized in Table 1, an image of a polyp is shown in Fig. 3 and the group averages are presented in Fig. 5. In the large intestine, the gender bios for polyposis is reversed ^35,37 and in the tested groups the average count of polyps in males is significantly higher than the one in females (2 factor ANOVA gender; F = 13.230, p < 0.001). More importantly, a significant reduction in the number of polyps after the administration of 1%FS2 (2 factor ANOVA FS2; F = 3.920,p = 0.049), yet no interaction between gender and FS2 (F = 1.566, p = 0.212) was observed. This is consistent with the effects of FS2 seen in the small intestine and suggests Apc signal regulation of FS2.

Figure 5. Total average counts in the colon for each group are shown. The error bars indicate the Standard Error from the Mean (SEM). The dotted and the solid lines indicated male and female animals respectively. Males displayed a significantly higher number of tumors over females, and a significant reduction of counts was observed between the control (H₂O) and the 1%FS2 treated animals.

Discussion

Dietary fiber consumption has been associated with a lower risk of human colorectal cancer.^40,41 Different mechanisms have been proposed to explain how fiber can protect colorectal cells from carcinogenesis, those including increased stool bulk and reduced stool transit time, formation of short-chain fatty acid by dietary fiber fermentation, and the reduction of secondary bile acid production.⁴² Fermentation production of short-chain fatty acids are suggested to promote a healthier gut microbiota ⁴³ and induce differentiation, arrest growth, and apoptosis of cells in the gastrointestinal tract .⁴⁴

Development of colorectal cancers is closely associated with mutations in the Apc gene, leading to increased transcription of genes involved in cell fate, proliferation and carcinogenesis.^5,45 Apc^MIN/+ mice are highly susceptible to spontaneous formation of intestinal adenomas and adenomatous polyposis.²¹

In the current study we demonstrated that FS2 has anticancer effects by increasing apoptosis and ROS generation in SW480. In Multiple Intestinal Neoplasia mice (Apc^MIN/+), treatment with FS2 did significantly reduce the number of adenomas developing in the small intestine and decreased the number of polyps formed in the colon in the animals. The finding that FS2 affected Apc-mutant cell line as well as Apc^MIN/+ mice's pathophysiology suggests an anti-tumor mechanism independent of the microbiota and fermentation products.

Previous studies of our group demonstrated that the apoptotic activity of FS2 is, at least in part, p53/Bax dependent, leading to increased cleavage of caspase 3 and 9.²⁹ In the present study we demonstrate that a significant increase in apoptosis caused by FS2 was observed in SW480 cells in which p53 are mutant.⁴⁶ Furthermore an increase in ROS formation was observed in SW480 cells treated with FS2. These results suggest that ROS could mediate p53-independent apoptosis in these cells. Similarly, previous studies demonstrated that FS2 reduces mitochondrial membrane potential, increasing ROS production in HCT116 cells.²⁹

In conclusion, these data strongly suggest that direct exposure of cancer cells to components of FS2 inhibit cancer cell growth and proliferation, presumably independent of metabolic products formed from FS2 fermentation in the large intestine. Future studies are required to elucidate the mechanism of a microflora independent FS2 activity in more detail.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We want to thank Dr. William Dove at the University of Wisconsin Madison, WI for supplying the Apc^Min/+ mice and his laboratory staff for providing assistance establishing the colony.

Funding

This works was supported by Matsutani Research Fund, R01CA90631 and 5P01CA16056 (T.O.).