Tumor suppression by resistant maltodextrin, Fibersol-2

Abstract

Resistant maltodextrin Fibersol-2 is a soluble and fermentable dietary fiber that is Generally Recognized As Safe (GRAS) in the United States. We tested whether Fibersol-2 contains anti-tumor activity. Human colorectal cancer cell line, HCT116, and its isogenic cells were treated with FIbersol-2. Tumor growth and tumorigenesis were studied in vitro and in vivo. Apoptotic pathway and generation of reactive oxygen species (ROS) were investigated. We discovered that Fibersol-2 significantly inhibits tumor growth of HCT116 cells by inducing apoptosis. Fibersol-2 strongly induces mitochondrial ROS and Bax-dependent cleavage of caspase 3 and 9, which is shown by isogenic HCT116 variants. Fibersol-2 induces phosphorylation of Akt, mTOR in parental HCT116 cells, but not in HCT116 deficient for Bax or p53. It prevents growth of tumor xenograft without any apparent signs of toxicity in vivo. These results identify Fibersol-2 as a mechanism-based dietary supplement agent that could prevent colorectal cancer development.

Keywords:

• apoptosis
• colorectal cancer
• Fibersol-2
• p53
• resistant maltodextrin
• reactive oxygen species

Abbreviations

BAX	=	Bcl-2-associated X protein
MFI	=	mean fluorescence intensity
mTOR	=	mammalian target of rapamycin
PARP	=	poly ADP reibose polymerase
ROS	=	reactive oxygen species

Introduction

Colorectal cancer is one of the most common malignant diseases of Western societies and its incidence appears to be strongly influenced by environmental factors.¹ Diets that are low in fat and high in fruits, vegetables, and fiber, are associated with a lower cancer risk.^1-3

Physical and chemical properties of dietary fibers have been extensively investigated using epidemiological methods but have not been confirmed conclusively.^4,5 It has been implicated that fermentation of dietary fiber into short-chain fatty acids subsequently decreases soluble bile acids in the large bowel.^5-8 Other possible mechanisms are an increase in fecal bulking, which could dilute carcinogenic components in the feces in transit time.^9-12 However, not all dietary fibers indicate the same biological effects on fermentation and fecal bulking.^12-15 Thus, biological activities of dietary fiber to lower colon cancer risk remains to be conclusive.

Resistant starch is insoluble and an undigested starch that reaches the colon undigested, just like dietary fiber.¹⁶ It is a potential source of fermentable substrate and decreases the fecal bile acids^17,18 and fecal pH,¹⁹ increases total fecal bulking ^18,19 and excretion of short-chain fatty acid.^18,19,20 Thus, resistant starch may have similar activities with dietary fiber to regulate fermentation and fecal bulking. Recent clinical studies have shown that the healthy volunteers could take resistant starch up to 6 times of the daily amount in a normal Western diet, indicating that resistant starch is possible alternative for dietary fiber.¹⁸

Distinct from resistant starch, Fibersol-2 is a soluble and fermentable dietary fiber made from corn starch. It is a nonviscous low-calorie bulking fiber and has been used with a variety of food applications. It contains numerous starch linkages that remain undigested by enzymes in the digestive tract. Resistant maltodextrin is not digested or absorbed in the human small intestine, and thus passes to the large intestine where it is fermented by the colonic bacteria producing short-chain fatty acids, lower the pH, gaseous byproducts and beneficial bacteria.⁵ Clinical studies have shown that postprandial blood concentration of glucose, insulin and serum lipids are decreased and fecal volume is increased after consumption of Fibersol-2.²¹ Continuous consumption of Fibersol-2 also decreases the risk factors of metabolic syndrome with improved glucose and lipid metabolism.²²

In the current studies, we investigated whether Fibersol-2 contains anti-tumor activity in cell culture and mouse model systems. We found that Fibersol-2 significantly increases mitochondrial reactive oxygen species (ROS). Isogenic cell lines of HCT116 variants indicate that cleavage of PARP and caspase 3 by Fibersol-2 is p53 and Bax dependent. Anti-tumor activity in vivo was demonstrated by the decreased tumor development in mouse xenograft. These results establish the usefulness of Fibersol-2 for the prevention human colorectal cancer.

Results

Fibersol-2 induces apoptosis and suppresses the anchorage independent HCT116 p53(-) cells growth

Inhibition of tumor growth by Fibersol-2 was explored by colony formation assay in soft agar. HCT116 and HCT116 p53(-) cells ²³ were plated in soft agar, and colony formation was observed. As shown in Figure 1, both HCT116 and HCT116 p53(-) cells formed colonies in 7 to 10 days. Diameter of colonies of HCT116 p53(-) cells was much larger than those of HCT116 cells. Significantly, when Fibersol-2 was provided, both of these cells did not grow in soft agar, indicating that Fibersol-2 directly inhibits tumorigenecity.

Figure 1. Colony formation in soft agar is strongly inhibited by Fibersol-2. Parental HCT116 and p53(-) cells (1 × 10⁴ cells/60 mm plate) were treated with 3% Fibersol-2 in soft agar culture. Colonies were photographed at day 10. Data were presented as representative of 3 independent experiments.

Anti-tumor activities of Fibersol-2 were studied by FACS/Annexin V analysis (Fig. 2). Isogenic cell lines, HCT116, HCT116 p53(-), HCT116 Bax(-) and HCT116 Puma(-),^24,25 were treated with PBS or Fibersol-2 (final concentration 1% or 2%) for 24 h. Apoptosis was determined by Annexin V/PI double staining. As shown in Fig. 2, apoptosis induced by Fibersol-2 (1%) was 30.4%, 19.4%, 19.9%, and 18.8%, and apoptosis induced by Fibersol-2 (2%) was 85%, 50.1%, 63.1%, and 88.8% in HCT116, HCT116 p53(-), HCT116 Bax(-) cells, and HCT116 Puma(-) cells, respectively. These results show that p53 or Bax, but not Puma, is essential for the maximal induction of apoptosis by Fibersol-2. Besides, HCT116 p53(-) and HCT116 Bax(-) cells still underwent lower levels of apoptosis, suggesting that FIbersol-2 can induce apoptosis in p53/Bax-independent manner.

Figure 2. Fibersol-2 induces apoptosis of Isogenic HCT116 cell lines. Parental and 3 different isogenic knockout HCT116 cell lines (p53(-), Bax(-), and Puma(-)) were treated with vehicle only (1 × PBS, control), 1% or 2% Fibersol-2 (in 1X PBS). After 24 h, cells were harvested and analyzed of apoptotic cell population by double staining with PtdIns/annexinV as described in M&M. Figure was representative of 3 independent experiments.

Fibersol-2 regulates cell survival signals

We next studied the biochemical analysis of pro-apoptotic proteins when cells are treated with Fibersol-2 (1% or 2%) for 24 hours (Fig. 3). Cleaved forms of PARP, caspase 3 and caspase 9 are detected in HCT116 and HCT116 Puma(-) cells, but not in HCT116 p53(-) and HCT116 Bax(-) cells. Together with the results of Annexin V staining of apoptotic cells (Fig. 2), these results indicate that cleavage of these proteins in HCT116 cells is essential for the maximal induction of apoptosis by Fibersol-2.

Figure 3. Fibersol-2 treatment activates both pro-apoptotic and anti-apoptotic signaling pathways. 1% and 2% Fibersol-2 (10 and 20 mg/ml in PBS) were added to culture media of parental and isogenic knockout HCT116 cell lines (p53(-), Bax(-), and Puma(-)) (A & B). After 24 h, total lysates were prepared and subjected to western blot analysis (30μg/sample). Doxorubicin (1 μM) was used as positive control to induce DNA damage signaling pathway (B).

Regulation of cell survival by mTOR/Akt pathway has been well illustrated.^26-28 Interestingly, treatment of HCT116 cells with Fibersol-2 induced phosphorylation of mTOR at Ser2481, Akt at Ser473 and p53 at Ser15 in dose dependent manner (Fig. 3B). In these experiments, doxorubicin was used as a positive control to induce p53 phosphorylation. Of note, phosphorylation of mTOR and Akt is p53-dependent since it is not observed in HCT116 p53(-) cells. Furthermore, phosphorylation of these proteins is not induced in HCT116 Bax(-) cells, although p53 is similarly phosphorylated in those cells. These results suggest that p53/Bax axis is important to induce phosphorylation of mTOR and Akt when exposed to Fibersol-2.

Fibersol-2 induces mitochondrial ROS

It has been extensively studied that Bax plays crucial roles for mitochondria-mediated apoptosis.²⁹ We studied whether Fibersol-2 regulates mitochondria homeostasis. We examined mitochondrial integrity and ROS production in HCT116 and HCT116 p53(-) cells (Fig. 4). JC-1 was used as a fluorescence marker of mitochondrial transmembrane potential (ΔΨμ) as previously described.^30,31 When mitochondria functions normally, the JC-1 concentrates inside mitochondrial membrane. A cytotoxic event that dissipates the mitochondrial membrane potential prevents the accumulation of the JC-1 dye in the mitochondrial matrix and thus, JC-1 remains as a monomeric form (green fluorescent) in cytoplasm. Fibersol-2 treatment induced increased JC-1 monomeric signals in HCT116, indicating that Fibersol-2 perturbs mitochondrial membrane potential. Basal levels of JC-1 signals were higher in HCT116 p53(-) cells, and they did not change after Fibersol-2 treatment in those cells, suggesting that mitochondria function is continuously perturbed in HCT116 p53(-) cells.

Figure 4. Fibersol-2 increases mitochondrial ROS of HCT116 and HCT116 p53(-) cells. Parental and p53(-) HCT116 were incubated in 2% or 4% Fibersol-2 containing cDMEM for 24 h, and then stained with JC-1 dye at 2.5 μg/ml for 10 min (upper panel). Mitochondrial ROS levels in parental and p53(-) HCT116 cells were detected by MitoSox (lower panel). Quantitative flow cytometry data were expressed as ΔMFI. Data were representative of 2 independent experiments.

Next, we measured levels of mitochondrial ROS with MitoSox, which are indicated by MFI (mean fluorescence intensity). Treatment with Fibersol-2 resulted in a high increment in MitoSox fluorescence intensity in HCT116 (3.3 fold), compared to HCT116 p53(-) (2.3 fold). These results indicate that treatment with Fibersol-2 induces perturbation of mitochondria homeostasis, such as change in mitochondrial membrane potential and increase in mitochondrial ROS.

Fibersol-2 decreases tumor development of HCT116 in xenograft study

Inhibition of tumor growth by Fibersol-2 was further investigated with HCT116 xenograft model. On day 7 when small size of tumors was observed, we started s.c. injection of Fibersol-2 (1% and 5% in water) with these mice, and tumor growth was measured at indicated day. As demonstrated in Fig. 5, both 1% and 5% of Fibersol-2 significantly decreased tumor growth during this time course. Tumor growth of xenograft of a breast cancer cell line, MDA-MB-468 cells, was also delayed, although this inhibition was not as effective as that seen in HCT116 cells (data not shown). Given the results showing that FIbersol-2 induces caspase-mediated apoptosis (Fig. 3), it is assumed that injected Fibersol-2 directly decreases tumor growth by activating apoptosis pathway.

Figure 5. Inhibition of tumor growth by Fibersol-2. HCT116 cells were transplanted into nude mice as described in M&M. 1% or 5% Fibersol-2 solution (in ddH2O) was injected (s.c.) around tumor. Tumor size was measured at indicated times.

These in vivo results demonstrate that Fibersol-2 contains a mechanism-based anti-tumor activity, and suggest that it can be considered for clinical application for colorectal tumors.

Discussion

It has been debated whether fiber consumption can reduce colorectal cancer incidence.^32,33 At least 2 possible mechanisms of how dietary fiber inhibits colorectal cancer development have been implicated; (a) it maintains digestive movement of the bowels to minimize the exposure to carcinogens, and (b) metabolites (e.g. butyrate) from bacterial fermentation in intestine inhibit tumor cell growth.³⁴ However, it has not been well explored whether dietary fiber directly affects cancer cell proliferation. The present study first describes about anti-cancer effects of specific dietary fiber (resistant maltodextrin), Fibersol-2. The treatment with Fiberso-2 resulted in apoptosis and reduction in tumor development in xenograft model.

Regulation of apoptosis by p53 has been extensively studied.³⁵ To understand the roles of p53 pathway in inducing apoptosis by Fibersol-2, we used isogenic colon cancer cell lines generated from HCT116 cells in which p53-associated genes are disrupted individually. Compared to the parental HCT116 cells, HCT116 p53(-) and HCT116 Bax(-) cells showed less apoptosis when treated with Fibersol-2, suggesting the Fiberso-2's apoptotic activities are at least in part p53/Bax dependent. We also observed that Fibersol-2 induced activation of caspase 3 and 9 in the parental HCT116 cells, but not in HCT116 p53(-) and HCT116 Bax(-) cells, indicating that cleavage of caspase 3 and 9 induced by Fibersol-2 is p53/Bax dependent. These results suggest that FIbersol-2's cytotoxicity is mediated by cleaved caspase 3 and 9.

We found that Fibersol-2 reduces mitochondrial membrane potential, causing mitochondrial malfunction, and increases mitochondrial ROS production (Fig. 4). These results, together with other biochemical analyses (Fig. 3), suggest a model that increase in levels of ROS activates p53, resulting in induction of Bax. Elevated Bax induces translocation of cytochrome c and Apaf-1, causing formation of apoptosome containing caspase 9.³⁶

Colony formation of HCT116 p53(-) cells in soft agar was strongly inhibited by Fibersol-2, showing p53 independent mechanism of cytotoxicity. Consistent with this model, HCT116 p53(-) cells underwent apoptosis when treated with Fibersol-2, although efficiency is lower compared to the parental HCT116 cells. These results support a hypothesis that Fibersol-2 can induce apoptosis by p53 dependent and independent manner. Puma is one of the transcriptional targets of p53 and it interacts with anti-apoptotic Bcl2 family member proteins, releasing pro-apoptotic proteins, Bax and Bak, leading to cell death through mitochondrial apoptosis pathway.³⁷ Our results indicating Puma-independent Fibersol-2's activity are consistent with the recent study showing stress-induced cell death by Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation.³⁸

It has been well illustrated that Akt/mTOR signaling is involved in cell proliferation and anti-apoptosis.^26-28 However, recent reports described that single or combination treatment of anti-cancer drugs induces apoptosis through ROS/Akt signaling in colorectal cancer cells.^39,40 In fact, Akt regulates tumor cell death induced by nutritional starvation and ROS production.⁴¹ In our experiments, although Fibersol-2 induced mitochondrial ROS in both HCT116 and HCT116 p53(-) cells (Fig. 4), phosphorylation of Akt/mTOR was observed only in HCT116 cells in which p53 is wild type (Fig.3B). Furthermore, phosphorylation of mTOR and Akt is p53/Bax-dependent since it was not observed in HCT116 p53(-) and Bax(-) cells. Therefore, our findings suggest that p53/Bax axis is involved in activation of mTOR/Akt pathway during Fibersol-2-induced CRC cell apoptosis, although the mechanism of Akt activation by p53 remains to be elucidated. Given that Fibersol-2 induces apoptosis of these isogenic HCT116 variants (Fig. 2), it is suggested that Fibersol-2 produces ROS and subsequent apoptosis in p53/Akt-dependent and independent manner. Doxorubicin-induced DNA damage strongly induced p53 phosphorylation, but not phosphorylation of Akt, suggesting that Fibersol-2-induced activation of Akt is not due to DNA damage.

In summary, our study first characterized the novel effect of dietary fiber on cellular growth and tumor development by in vitro and in vivo experiments using colorectal cancer cell line. In addition, on-going comparison study between Fibersol-2 and other commercial dietary fibers shows that Fibersol-2 has more suppressive effects than the other on the growth of HCT116 cells (in progress). It has been shown that approximately 10% of Fibersol-2 is digested and absorbed in the small intestine^42,43 , therefore, it is suggested that this fraction of Fibersol-2 induces anti-tumor activity after circulation in the blood. On the basis of our results, a protocol of the Phase II clinical trial for the high risk colorectal cancer patients has been just approved at Roswell Park Cancer Institute, in which oral administration of Fibersol-2 will be studied.

Further biochemical analysis using Fibersol-2 fractions will contribute to a better understanding how Fibersol-2 directly suppresses cancer cell growth. Finding from these studies can assist the development of an anti-tumor adjuvant, and potential application against human colorectal cancer.

Materials and Methods

Cells and reagents

HCT116 (human colorectal carcinoma) cells were purchased from ATCC (CCL^-247, Manassas, VA), HCT116 p53(-), HCT116 Bax(-), and HCT116 Puma(-) cells were kindly provided by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD) who had originally established these cell lines. All of these cells were grown in MoCoy's 5a media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin (100 units/ml, Invitrogen). Fibersol-2 was provided by Matsutani America (Itasca, IL), and solubilized in 1X PBS for cells or water (w/v) (for mouse feeding). Mitochondrial superoxide indicator (MitoSOX) was purchased from Invitrogen. JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbeznimidazolylcarbocyanine iodide) was obtained from eBioscience (San Diego, CA). Doxorubicin was purchased from Sigma-Aldrich (St. Louis, MO).

Soft Agar Assay for anchorage independent cell growth

DMEM supplemented with 10% calf serum and 0.4% agarose containing 1 × 10⁴ of either HCT116 and HCT116 p53(-) cells were plated on DMEM supplemented with 0.5% agar, 10% calf serum and 3% of Fibersol-2. Cell culture was maintained for 7 to 10 days and colony formation was studied under a microscope. Photographs were taken under 40× magnifications using a SPOT Insight mosaic microscope camera (SPOT Imaging Solutions, Sterling Heights, MI) attached to Leica DM IRB microscope (Buffalo Grove, IL).

Flow cytometry for apoptosis analysis

HCT116, HCT116 p53(-), HCT116 Bax(-), and HCT116 Puma(-) cells were treated with or without Fibersol-2 (1% or 2%). After 24 h culture, the rate of apoptotic cells were examined after double staining with Annexin V-FITC/Propidium Iodide (PI) 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).

Western blot assay

Cells were treated Fibersol-2 (1% or 2%) or Doxorubicin (1 μM) for 24 h, then lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.6),150 mM NaCl, 1 mM EDTA (pH 8.0), 20 mM NaF, 1 mM Na₃VO₄, 1% NP40, 0.5 mM dithiothreitol) in the presence of protease–inhibitors (leupeptin, aprotinin, and PMSF, 10 mg/ml, respectively). Total cell lysate (30 μg) was loaded in 15 or 6% SDS-PAGE, and transferred to a PVDF membrane (Immobilon-P, Millipore, Billerica, MA). Primary antibodies used in this study were anti-Akt, anti-mTOR, anti-PARP, anti-cleaved caspase 3 (Asp175), anti-cleaved caspase 9 (Asp330) (Cell signaling Technology, Danvers, MA), and anti-p53 (Santa Cruz Biotechnology, Santa Cruz, CA). Also specific anti-phosphorylation antibodies were used against phospho-Akt (Ser473), phosphor-mTOR (Ser2448), and phosphor-p53 (Ser15) (Cell signaling). Actin was detected as internal loading control.

Measurement of mitochondrial membrane potential and ROS

HCT116 and HCT116 p53(-) cells were treated with 2% or 4% Fibersol-2 for 24 h. To determine mitochondrial membrane potential (ΔΨm) , cells were incubated with JC-1 (2.5 μg/ml) for 10 mim at room temperature and then analyzed as described.³¹ For measurement of mitochondrial ROS, cells were cultured in media containing MitoSox (5 μM) for 10 min at 37°C. Single cell suspensions were quantitatively analyzed by flow cytometry. Data were expressed as histogram for Green (FL1, JC-1) or Red fluorescence (FL2, MitoSox), indicated by MFI (mean fluorescence intensity).

Tumor formation in nude mice

Female athymic nude mice (n = 10) were purchased from Jackson Lab (Bar Harbor, Maine), and housed in a pathogen-free facility of Roswell Park Cancer Institute. A total 5 × 10⁶ HCT116 cells were subcutaneously (s.c.) injected into the flanks. Fibersol-2 was solubilized in water (1% and 5%) and autoclaved. When small size of tumors was developed (200 ∼ 400 mm³), tumor was s.c. injected with Fibersol-2. Tumor growth was measured at day 13, 15, 20, 26, 30 and 33 after injection and tumor volume was determined using the equation:= (width)² × length/2. These procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Roswell Park Cancer Institute.

Acknowledgment

We thank all the members of the Ouchi Laboratory for helpful discussion. We particularly thank Dr. Bert Vogelstein at Johns Hopkins University for providing us with isogenic HCT116 cell lines.

Author Contributions

E.Y.S., M.O., S.C.S., S.L.O. and T.O. planned and generated all the data. E.Y.S, D.R., K.S., and T.O. discussed the results, and E.Y.S. and T.O. wrote the paper.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work is supported by Matsutani America Cancer Research Fund, R01CA79892, R01CA90631, and 5P30CA16056 from National Institutes of Health, and Breast Cancer Research Grant from Susan G. Komen Foundation.