| Background and aimsColorectal cancer (CRC) is one of the most common cancers terribly endanger human life worldwide. CRC is the second leading cause of cancer-related deaths in North America and Europe. As the socio-economic development, lifestyle and eating habits change recently, the morbidity and mortality of CRC in China has an increasing trend. The therapeutic strategy for CRC is a comprehensive method including surgery, chemotherapy, radiotherapy, biological therapy and so on. But the whole cure rate of CRC was only about 50% in spite of using all these methods and the long-term survival rate and the quality of life were not satisfied. Therefore, emphasis has been focused on chemoprevention of CRC. Suppression of the carcinogenic process by use of pharmacological or natural agents is the cornerstone of chemoprevention. CRC has a natural history of transition from a precursor lesion, adenomatous polyp to cancer, which spans over 10 to 15 years providing an extended opportunity for intervention and cancer prevention. Recently, emphasis has been on a variety of clinical and basic studies of chemoprevention using naturally occurring substances that are found in normal diets, since they might provide useful strategies to inhibit CRC with minimal toxicity.Epidemiological evidence suggests that high intake of horseradish, cabbage, cauliflower and other cruciferous vegetables has been associated with lower risk of prostate cancer, lung cancer, bladder cancer, esophagus and colorectal cancer in the human population. The seeds of Sinapis alba Linn. (commonly called white or yellow mustard) have been used as a spice and as an herbal in China, and is included in the Pharmacopoeia of the People's Republic of China. Mustard seed is believed by doctors of traditional Chinese medicine to have the action of reliving dyspnea and cough by eliminating cold phlegm, reducing nodulation, and reliving pains by removing the obstruction of collaterals. Recently mustard seed and its components have been demonstrated to possess anticancer activity. Mustard seed oil significantly decreased tumor incidence and number in DMBA-induced transplacental and translactational carcinogenesis in Swiss albino mice. The ethanolic extract of mustard seeds was shown to reduce tumor incidence and burden in 7,12 dimethylbenz (a) anthracene (DMBA)-induced skin papillomagenesis in Swiss albino mice. Gagandeep et al. reported the chemopreventive efficacy of mustard seed mixed diets against benzo(a)pyrene[B(a)P]-induced forestomach tumorigenesis and 3-methylcholantrene (MCA)-induced tumorigenesis in the uterine cervix of mice. However, there is limited study regarding the use of mustard seed to prevent CRC, and the anticancer mechanism of mustard seed is unclear.In recent years, more and more attentions are paid on the relationship between free radicals and tumor. Findings showed that free radicals not only cause damage to cells, leading to changes of cell structure and function, but also played an important role in carcinogenic and cancerigenic process. Free radicals are atoms with non-paired electrons, atoms, molecules or ions, which are essential for many important reactions of normal life activities, such as involving the synthesis of various biologically active substances, detoxification reactions, and the process of phagocytes to kill bacteria and so on. But when external toxic chemical and physical factors such as radiation, toxins, chemical carcinogens and other effects on the body, the balance between production and scavenging of free radicals will be disturbed, resulting in extensive damage effect. Free radicals can cause lipid peroxidation, change protein, enzyme activeness, damage mitochondrion, attack DNA and induce gene mutation. Biological organism mainly prevent radical injury through scavenging enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) etc. They respectively act on radical chain reaction in different links in vivo to scavenge free radicals. SOD dismutates the pair of superoxide anions by oxidizing one to oxygen and reducing the other to hydrogen peroxide. Hydrogen peroxide is converted into water and oxygen by CAT. GSH-Px can both clear hydrogen peroxide and lipid hydroperoxide. MDA is a metabolic product of lipid peroxidation. The level of MDA is often used as a marker of oxidative damage. Above indicators can comprehensively response oxidation and antioxidant status of organism.The animal model of tumor is an important tool for research of tumor etiology, prevention, early diagnosis and treatment. The most commonly used model for CRC takes advantage of the organotropism of the colon carcinogens, 1,2-dimethylhydrazine (DMH) and its metabolite, azoxymethane (AOM). AOM offers advantages over DMH, including enhanced potency and greater stability in dosing solution. It is a strong oxidant that can cause peroxidation to cell membranes and DNA. The acquired mutations to DNA, results in cell proliferation thereby, leading to colon carcinogenesis. Experimental colorectal cancer induced by AOM in mice is a prolonged multistage process, bearing many of the same cell kinetics, histopathological and molecular characteristics of tumorigenesis that mimics human colorectal cancer.Based on the above background, the purpose of this study is to investigate the chemopreventive effects of mustard seed on azoxymethane (AOM)-induced colorectal tumor in mice and to explore its anticancer-related mechanisms. The status of proliferation and apoptosis of colorectal tumor induced by AOM in mice will be detected by immunohistochemistry. The effects of mustard seed on the level of oxygen free radicals and the activity of antioxidant enzymes in mice are to be observed. From the angle of free radical reactions in tumorigenesis, the prevention mechanism of mustard seed on colorectal cancer is to be investigated. This study will provide experimental evidence and theoretical guidance for the mustard seed used in the chemoprevention of colorectal cancer in future.Materials and Methods1. A total of 60 female mice of Kunming species(body weight 18-20g) were randomly divided into 4 groups of 15 each:AOM alone, AOM+5%MS, AOM+10%MS, and the untreated control groups. Colorectal tumorigenesis was induced by injecting 10 mg/kg.bw of AOM, intraperitoneally, once in a week for three weeks. Different doses of MS were given in diet during the study.2. Observe the-general condition of mice, such as eating and drinking, nutrition, weight, physical activity, coat color, gloss change, stool, etc.36 weeks after initiation, the mice were sacrificed and blood sample were collected. The large intestine were isolated and flushed with ice-cold normal saline.3. The site, size, and number of tumors in each group were recorded. The incidence, the average number and the inhibitory rate of colorectal tumor in each group was determined. Other organs were observed with or without metastasis.4. Tumor tissues were conventional fixed and dehydrated, paraffin-embedded sections, H.E staining to determine the histological type. 5. Immunohistochemical staining to detect the expression of PCNA protein in tumor tissue and calculate tumor cell proliferation index (PI).6. TUNEL staining to detect the apoptosis of tumor tissue and calculate tumor cell apoptosis index (AI).7. Colorimetric assay for activities of antioxidant enzymes (SOD, CAT, GSH-PX) and levels of lipid peroxidation product malondialdehyde (MDA) in serum of mice in each group.Results1. At the termination of experiment, the mice in AOM model group showed weight loss, anorexia, hair removal, bloody stool, and 2 mice appeared ascites. The body weight in AOM model group were significantly lower than normal control group, with significant differences (P<0.05). The body weight in MS intervention group were increased compared with AOM model group, with significant differences (P<0.05). The body weight in 5%MS and 10%MS intervention group were no significant difference(P>0.05).2. No tumor was found in normal control group.The colorectal tumor incidence, as compared to AOM model group (86.7%), was reduced to 60.0% by the 5% diet of mustard seeds and to 41.7% by the 10% diet of mustard seeds. The tumor incidence of the three groups was statistically significant difference(χ2=6.607, P=0.048<0.05), with the highest incidence in AOM model group, followed by 5%MS intervention group,10%MS intervention group minimum.3. Tumor burden (tumors/mouse) was 2.20±1.21 in the AOM model group, whereas it was reduced to 1.07±1.10 (P<0.05, Compared with model group) by the 5% dose of mustard seeds, and 0.67±0.89 (P<0.05, Compared with model group) by the 10% dose of mustard seeds. Tumor inhibition rate was 51.4% in 5%MS intervention group and 69.5% in 10% MS intervention group. 4. PI in AOM model group,5%MS and 10%MS intervention group were: 59.9±4.4,41.7±4.9 and 32.0±3.9, and the tumor cell proliferation index in 10%MS intervention group was significantly lower than the AOM model group and 5% MS intervention group, with significant differences (P<0.05).5. AI in AOM model group,5%MS and 10%MS intervention group were: 6.9±1.4,9.3±1.5and15.0±2.4, and the tumor cell apoptosis index in 10%MS intervention group was significantly higher than the AOM model group and 5% MS intervention group, with significant differences (P<0.05).6. Serum SOD, CAT and GSH-PX activities in AOM model group were significantly lower than the normal control group, with significant differences(P <0.05). Serum SOD, CAT and GSH-PX activities in 10%MS intervention group was significantly higher than the AOM model group and 5% MS intervention group, with significant differences (P<0.05).7. Serum MDA content in AOM model group was significantly higher than the normal control group, with significant differences(P<0.05). Serum MDA content in 10%MS intervention group was significantly lower than the AOM model group and 5% MS intervention group, with significant differences (P< 0.05).Conclusions1. The mustard seeds possess chemopreventive activity against colorectal cancer induced by AOM in mice, and can reduce the incidence of tumor and average number of tumor. The concentration of mustard seed is higher, and the effect of prevention of colorectal cancer is stronger.2. The mustard seeds can reduce the expression of PCNA in colorectal tumor tissue and inhibit tumor cell proliferation and induce apoptosis of tumor cells.3. The mustard seeds can enhance the activities of SOD,CAT and GSH-PX in serum, and decrease serum lipid peroxidation products MDA during the AOM carcrnogenic process.4. The prevention mechanism of mustard seeds on colorectal cancer is related with its antioxidant activity, by increasing the body's antioxidant enzyme activity, enhancing antioxidant capacity, reducing free-radical damage to cells, preventing mutations and tumor. |
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