Resistant starch is as common soluble fiber that escapes digestion in the small intestine and can regulate intestinal function, metabolism of blood glucose and lipids, and may prevent tumorigenesis of gastrointestinal cancer. Epidemiology and other evidence have suggested that resistant starch may prevent colon cancer development. The aim of the current study was to explore the ameliorative effects and potential mechanisms of resistant starch in the tumorigenesis of colon tumors induced by dimethylhydrazine in C57BL/6 mice. Western blot analysis, ELISA, microscopy, immunofluorescence and immunohistochemistry were used to analyze the efficacy of resistant starch on the metabolic balance in the colon and tumorigenesis of colon tumors. The results demonstrated that a diet containing resistant starch decreased the animal body weight and reduced free ammonia, pH and short chain fatty acids in feces compared with mice that received a standard diet. Resistant starch reduced the incidence of colon tumors and suppressed the expression of carcinogenesis-associated proteins, including heat shock protein 25, protein kinase C-d and gastrointestinal glutathione peroxidase in colon epithelial cells compared with standard starch and control groups. Colon tumor cells proliferation and dedifferentiation were significantly decreased by a resistant starch diet. The results also demonstrated that resistant starch increased the apoptosis of colon tumor cells through regulation of apoptosis-associated gene expression levels in colon tumor cells. Oxidative stress and endoplasmic reticulum stress were upregulated, and elevation eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor-4 and secretase-β expression levels were increased in the resistant starch diet group. Additionally, the activity of eIF2α and PERK were increased in colon tumor cells from mice that had received resistant starch. Increasing DNA damage-inducible transcript 3 protein (CHOP), binding immunoglobulin protein (BIP) and caspase-12 expression levels upregulated by resistant starch diet may contribute to the resistant starch-induced apoptosis of colon tumor cells induced by 1,2-dimethylhydrazine.
Colon cancer is one of the most common gastrointestinal tumors; it is the second most common cancer in women and third in men worldwide (
Resistant starch is widely present in carbohydrate starch material and has miscellaneous effects in colon metabolism (
Increasing apoptosis of tumors cells has benefits for prevention and treatment of colon cancer through regulation of the expression of apoptosis-associated proteins (
The current study investigated the anticancer effects and potential mechanisms of resistant starch in the tumorigenesis, formation and development 1,2-dimethylhydrazine-induced colon cancer. The colon physiological functions of experimental mice were analyzed following consumption of a diet containing resistant starch. Notably, this analysis investigated whether resistant starch induces apoptosis of colon tumor cells following treatment with 1,2-dimethylhydrazine.
This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (
A total of 20 C57BL/6 mice, 6–8 weeks old, were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and housed in a temperature-controlled room (25±1°C) with artificial 12/12 h light/dark cycle. All mice could access water containing 1,2-dimethylhydrazine (3 mg/kg) to induce colon cancer. The incidence of colon tumor induced by 1,2-dimethylhydrazine was calculated by histopathology as described in immunohistochemistry assay. Experimental mice were divided into two groups (n=10/group) with free access to a regular diet (5 mg/kg) or a resistant starch diet (5 mg/kg). All mice were sacrificed for further analysis on day 120.
Ammonia in experimental mice was measured using ionization constant. pH was determined by pH meter (Mettler). Short chain fatty acids were analyzed using High Performance Liquid Chromatography (Takara, Tokyo, Japan).
Colon epithelial cells and colon tumor cells were isolated from experimental mice and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KgaA, Darmstadt, Germany). Cells were cultured at 37°C and 5% CO2.
RNA was reverse transcribed into cDNA at 42°C for 2 h using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. PCR amplification had preliminary denaturation at 94°C for 2 min, followed by 45 cycles of 95°C for 30 sec; the annealing temperature was reduced to 56.8°C for 30 sec and 72°C for 10 min. The reaction volume was a total of 20
Colon tumor cells were homogenized in a radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KgaA) and centrifuged at 6,000 × g at 4°C for 10 min. Protein concentration was measured with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). A total of 10
Colon tumor cells (1×106) were transfected with 100 pmol of siRNA targeting eukaryotic translation initiation factor 2α (eIF2α) with siRNA-vector as control (both from Applied Biosystems; Thermo Fisher Scientific, Inc.) using a Cell Line Nucleofector kit L. (Lonza Group, Ltd., Basel, Switzerland). Cells were cultured in 2.5 ml DMEM containing 10% FBS 6-well plates for 24 h. All siRNAs were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) including siRNA-eIF2α (eIF2α, L-015389) or siRNA-vector (scramble, D-001810). Cells were used for the subsequent assays after 48-h transfection.
Colon tumor cells isolated from experimental mice and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KgaA) at a 37°C humidified atmosphere of 5% CO2. Cell colonies growing on Matrigel® were loosely detached by dispase treatment for 5 min, washed 3 times with PBS. Cells were resuspended in DMEM medium containing 20% FBS. Cells were maintained on 1% agar-coated and allowed to differentiate for another 18 days. Cells were then fixed with 10% formalin for 1 h at 37°C. Next following stained with the 60% Oil Red O in isopropanol as working solution for 10 min. The proportion of Oil Red O-positive cells was determined by counting stained cells under a light microscope.
Eukaryotic translation initiation factor 2-α kinase 3 (PERK) activity in colon tumor cells was analyzed by recombinant glutathione S-transferase-PERK (536-1,116 amino acids) with 6-His-full-length human eIF2α as a substrate (
Immunohistochemistry and immunofluorescent staining were performed according to the standard procedures (
Cells (1×107) were collected from the experimental mice, and fixed with 70% ethanol for 2 h at 30°C. Fixed cells were rehydrated in PBS for 5 min and incubated in RNase A (1 mg/ml) for 30 min at 37°C. The cells were then subjected to PI/RNase staining followed by flow cytometric analysis using a FACScan instrument (Becton Dickinson, Mountain View, CA, USA) and Cell Quest software (Becton Dickinson).
Colon tumor cells were isolated from experimental mice and trypsinized and collected for apoptosis analysis. The cells were adjusted to 5×106 cells/ml with phosphate-buffered saline, labeled with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) using an Annexin V-FITC kit, and analyzed with a FACScan flow cytometer (both from BD Biosciences, Franklin Lakes, NJ, USA). The treatments were performed in triplicate, and the percentage of labeled cells undergoing apoptosis in each group was determined and calculated using FCS Express™ 4 IVD software 1.0 (De Novo Software, Glendale, CA, USA).
All data were presented as mean + standard error with three independent experiments. Statistical significance was analyzed using two tailed Student's t-test between groups. Unpaired data was analyzed by variance. P<0.05 was considered to indicate a statistically significant difference.
In order to investigate the benefits of resistant starch diets for metabolism of experimental mice, body weight and metabolic characteristic of colon tissues were analyzed. A resistant starch diet decreased body weight compared with a regular diet (
Anti-tumorigenesis efficacy of resistant starch was investigated in colon tissues induced by 1,2-dimethylhydrazine. Results showed that resistant starch suppressed tumor formation in experimental mice treated by 1,2-dimethylhydrazine (
Tumor cell proliferation and differentiation has a vital role in tumorigenesis. Thus, the effect of resistant starch on colon tumor cell proliferation and differentiation was determined. Colon tumor cell proliferation was suppressed by resistant starch compared with the control group in 1,2-dimethylhydrazine-induced mice (
The anti-apoptosis effects of resistant starch were also analyzed in experimental mice with 1,2-dimethylhydrazine-induced colon tumors. The resistant starch diet promoted apoptosis of colon tumor cells (
Changes of oxidative stress in colon tumors and colon epithelial cells was investigated. Resistant starch diets reduced the mRNA and protein levels of SOD and GSH in colon tumor cells compared with mice that received the control diet (
The effect if resistant starch on ER stress and its potential mechanism in inhibition of tumor cells growth was investigated. The resistant starch diet increased the expression levels of eIF2α, ATF-4 and secretase-β (BACE1) in colon tumor cells compared with cells from control mice (
Colon cancer is one of the most common gastrointestinal tumors, with highly invasive ability characterized by rapid invasion of lymphatics, flow transfer and local invasion (
Tumorigenesis mechanisms are crucial for initiation and progression of tumor formation (
Proliferation and differentiation of colon tumor cells contribute to tumor migration and invasion mediated by molecular signaling through effector pathways (
Induction of apoptosis by tumor therapeutic agents is essential for cancer therapy (
Autophagy is a cellular progression of materials conversion that promotes the obliteration of metabolic precursors (
Studies have indicated that ER stress is associated with apoptosis of colon tumor cells (
In conclusion, resistant starch improved bowel function, and the outcomes indicated that the resistant starch diet improved body weight and the metabolic characteristic of colon tissues. The results indicated that resistant starch inhibited tumorigenesis in the colon by promoting apoptosis and autophagy by upregulation of ER stress-dependent PERK activity, which mediated by eIF2α in colon tumor cells from mice induced by 1,2-dimethylhydrazine. These investigations indicated that resistant starch prevents tumorigenesis of colon tumors induced by dimethylhydrazine via regulation of an ER stress-mediated mitochondrial apoptosis pathway, which may useful in the future for the prevention of tumorigenesis of colon tissues.
The authors declare that they have no competing interests.
Effects of resistant starch on body weight and metabolic characteristic of colon tissues. (A) Body weight of experimental mice fed with resistant starch after induced by 1,2-dimethylhydrazine. Resistant starch improves weight of (B) proximal colon and (C) distal colon compared to regular diets. (D) Effects of resistant starch on digesta weight in caecum, proximal colon and distal colon in experimental mice induced by 1,2-dimethylhydrazine. (E) Digesta pH in caecum, proximal colon and distal colon in experimental mice fed with resistant starch after induced by 1,2-dimethylhydrazine. Effects of resistant starch on (F) free ammonia, (G) pH and (H) SCFA in faeces in mice fed with resistant starch. *P<0.05 and **P<0.01. SCFA, short chain fatty acids.
Effects of resistant starch on tumorigenesis in colon tissues induced by 1,2-dimethylhydrazine. (A) Resistant starch inhibits tumor formation in experimental mice treated by 1,2-dimethylhydrazine. (B) Effects of resistant starch decreases incidence of colon tumor in a mouse model induced by 1,2-dimethylhydrazine. (C) Gene and (D) protein expression levels of HSP25, PKC-d and GI-GPx in colon epithelial cells in experimental mice fed with resistant starch after induced by 1,2-dimethylhydrazine. Effects of resistant starch on (E) gene and (F) protein expression levels of c-myc, Ras and p53 in colon epithelial cells. (G) Resistant starch diet inhibits formation of microtubule and tumor vessel in colon tissues in experimental mice. (H) Resistant starch diet inhibits protein express levels of MAT-1 and NRP-2 in colon tissues in experimental mice. **P<0.01. HSP25, heat shock protein 25; PKC-d, protein kinase C-d; GI-GPx, gastrointestinal glutathione peroxidase; MAT-1, CDK-activating kinase assembly factor MAT1; NRP-2, neuropilin-2 (magnification, ×40).
Effects of resistant starch on inhibition of colon tumor cells proliferation and differentiation. (A) Resistant starch diet inhibits colon tumor cells proliferation in colon tissues induced by 1,2-dimethylhydrazine. Effects of resistant starch on (B) gene and (C) protein expression levels of PCNA, claudin 1 and claudin 2 in colon tumor cells. (D) Resistant starch diet inhibits colon tumor cells differentiation in colon tissues induced by 1,2-dimethylhydrazine. Resistant starch diet blocks (E) gene and (F) protein expression levels of mTOR and HK-II in colon tumor cells. (G) Resistant starch diet arrests S phase of colon tumor cells in experimental mice. (H) Long-term survival rate of experimental mice between resistant starch diet and standard diet groups determined by Kaplan-Mrier. **P<0.01. PCNA, proliferating cell nuclear antigen; mTOR, mechanistic target of rapamycin kinase; HK-II, hexokinase-2 (magnification, ×40).
Resistant starch diet induces apoptosis of colon tumors through mitochondrial apoptotic pathway. (A) Resistant starch diet promotes apoptosis of colon tumor cells in mice treated by 1,2-dimethylhydrazine. (B) Effects of resistant starch diet on mitochondria damage in colon tumor cells in experimental mice (magnification, ×100). Resistant starch diet increases pro-apoptosis (C) gene and (D) protein expression levels of cleaved caspase-3 and caspase-9 in colon tumor cells induced by 1,2-dimethylhydrazine. Resistant starch diet increases anti-apoptosis (E) gene and (F) protein expression levels of p53 and Bcl-2 in colon tumor cells induced by 1,2-dimethylhydrazine. Resistant starch diet promotes expression levels of Apaf-1 and Bad in colon tumor tissue in mice treated by 1,2-dimethylhydrazine determined by (G) immunohistochemistry (tissue) and (H) immunofluorescence (cells) (magnification, ×40). **P<0.01. Bcl-2, apoptosis regulator Bcl-2; Apaf-1, apoptotic protease-activating factor 1; Bad, Bcl-2-associated agonist of cell death.
Resistant starch diet increases oxidative stress in colon tumors through regulation of autophagy progression. Effects of resistant starch on (A) mRNA and (B) protein levels of SOD and GSH in colon tumor cells. Effects of resistant starch on (C) mRNA and (D) protein levels of SOD and GSH in colon epithelial cells. (E) Resistant starch upregulates AMPK activity in colon tumor cells. (F) Resistant starch upregulates expression levels of DDIT3 and Beclin 1 in colon tumor cells in mice treated by 1,2-dimethylhydrazine. Resistant starch diet promotes (G) mitophagy and (H) reticulophagy of colon tumor cells in mice treated by 1,2-dimethylhydrazine (magnification, ×40). **P<0.01. SOD, superoxide dismutase; GSH, glutathione synthetase; AMPK, AMP-activated protein kinase; DDIT3, DNA damage-inducible transcript 3 protein; MAP1LC3A, microtubule associated protein 1 light chain 3 α; TOMM20, translocase of outer mitochondrial membrane 20; Neo (Nase), 5′-Nase-ALPase.
Resistant starch diet regulates endoplasmic reticulum stress-dependent PERK activity through upregulation of eIF2α phosphorylation in colon tumor cells. (A) Resistant starch diet increases expression levels of eIF2α, ATF-4 and BACE1 in colon tumor cells. (B) Effects of resistant starch diet on phosphorylation levels of eIF2α and PERK in colon tumor cells in mice treated by 1,2-dimethylhydrazine. (C) Effects of resistant starch diet on activity of eIF2α and PERK in colon tumor cells in mice treated by 1,2-dimethylhydrazine. (D) Resistant starch diet increases expression levels of CHOP, BIP and caspase-12 in colon tumor cells in mice treated by 1,2-dimethylhydrazine. (E) Effects of DReIF2α on PERK activity in colon tumor cells in mice induced by 1,2-dimethylhydrazine. (F) eIF2α knockdown inhibits resistant starch-suppressed ATF-4 and BACE1 expression levels in colon tumor cells in mice fed with resistant starch. (G) Knockdown of eIF2α suppresses expression levels of CHOP, BIP and caspase-12 in colon tumor cells in mice fed with resistant starch. (H) Knockdown of eIF2α inhibits resistant starch-induced apoptosis of colon tumor cells isolated from mice fed with resistant starch. **P<0.01. eIF2α, eukaryotic translation initiation factor 2α; ATF-4, activating transcription factor-4; BACE1, secretase-β; p, phospho; PERK, eukaryotic translation initiation factor 2-α kinase 3; CHOP, DNA damage-inducible transcript 3 protein; BIP, binding immunoglobulin protein; AMPK, AMP-activated protein kinase; DReIF2α, downregulation of eIF2α.
Primer sequences for RT-qPCR.
Gene name Sequences |
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HSP25 |
F: 5′-ATCGAGATCTAATGGAGCCAGGGGAGGCG-3′ |
R: 5′-ATCGAGATCTGGAGAAGGCGGAGGGCGCGG-3′ |
PKC-d |
Forward: 5′-GCATCCTCTTCAGTTACGTCC -3′ |
Reverse: 5′-AAGAGAGCTTCCGTAAGGCG-3′ |
GI-GPx |
Forward: 5′-GAGGATATTTCGTGCCGCGC-3′ |
Reverse: 5′-GGAAGCTCCTGAAGATCTGT-3′ |
c-myc |
Forward: 5′-ATTGGGACAGCTTGGATCAC-3′ |
Reverse: 5′-AGTCACACGTCATCGACACC-3′ |
Ras |
Forward: 5′-CGCATCCTCAAAGGAGACATTCC-3′ |
Reverse: 5′-CACATCGAGGTGAACGGGAGTAAG-3′ |
p53 |
Forward: 5′-AAGGATCCTCAGTCTGAGTCAGGCC-3′ |
Reverse: 5′-ACCACCATCCACTACAACTAC-3′ |
PCNA |
Forward: 5′-AGCACGGTAACGTAGGGTGT-3′ |
Reverse: 5′-CATTGGAGGCATAAGCTG-3′ |
Claudin1 |
Forward: 5′-AGAACCAGTGAGCCTGATACATACAG-3′ |
Reverse: 5′-GCAACTCCATCGGCCTTTCCTACCAG-3′ |
Claudin2 |
Forward: 5′-GGAGTAGAAGTCCCGCAGGAT-3′ |
Reverse: 5′-AGGCCTCCTGGGCTTCAT-3′ |
m-TOR |
Forward: 5′-CGTACATGTCAGCCAGCTTC-3′ |
Reverse: 5′-TGGAGGAATTCTTGCTTTGC-3′ |
HK |
Forward: 5′-GACGCAATCAATGTTTACTCG-3′ |
Reverse: 5′-TATTTGGTTGGTCAGCACAGG-3′ |
Caspase-3 |
Forward: 5′-TTGAGGTAGCTGCACTGTGG-3′ |
Reverse: 5′-GGGCGTGTTTCTGTTTTGTT-3′ |
Caspase-9 |
Forward: 5′-CCAACCAAATGAAGCCAAGT-3′ |
Reverse: 5′-GCCCTTGCCTCTGAGTAGTG-3′ |
CPI |
Forward: 5′-GGAACACCTCGCTCTCCA-3′ |
Reverse: 5′-GGGATTCCCTGGACCTAAAG-3′ |
Bcl-2 |
Forward: 5′-CGTCTTCAGAGACAGCCAGGAG-3′ |
Reverse: 5′-TGAACCGGCATCTGCACAC-3′ |
SOD |
Forward: 5′-TTTGCCAGCAGTCACATTGC-3′ |
Reverse: 5′-GTACCAGTGCAGGTCCTCAC-3′ |
GSH |
Forward: 5′-CCGATCCAATCTGTTCTGGT-3′ |
Reverse: 5′-CCAGGGCTTTTCAAAAATGA-3′ |
β-actin |
Forward: 5′-AGCCTTCTCCATGGTCGTGA-3′ |
Reverse: 5′-CGGAGTCAACGGATTTGGTC-3′ |