Tissue zinc levels in precancerous tissue in the gastrointestinal tract of azoxymethane (AOM)-treated rats

Alterations in tissue zinc levels have been documented in patients with gastrointestinal tract malignancies and more frequently, in those with colonic cancer. However, the precise role of tissue zinc in carcinogenesis is not well elucidated. This study, using a well-established colon cancer model in rats, was designed to investigate the relationship of tissue zinc to the carcinogenic process. The aim was to examine tissue zinc levels in the preneoplastic tissues and to study the changes that occur during transition of mucosa from normal to preneoplastic state. Six-week old rats were given a single dose subcutaneous injection of azoxymethane (AOM) (30mg/kg body weight) and sacrificed after 1, 2, 5, and 9 months of the treatment. Plasma zinc levels showed a significant decrease (p<0.05) at 9 months compared with controls. Tissue zinc levels showed a significant decrease in the large intestine at 1 and 2 months (p<0.05) and at 5 and 9 months (p<0.01), in the small intestine at 2, 5, and 9 months (p<0.05), and in the stomach at 5 and 9 months (p<0.05). The maximum percent decrease (45%) in tissue zinc was observed in the large intestine at 9 months. Tissue copper zinc super oxide dismutase (CuZnSOD) activity was assessed in the body of the stomach, small intestine, and large intestine and compared with the control group. There was a significant fall in CuZnSOD levels in the small intestine at 9 months (p<0.05) and in the large intestine at 5 and 9 months (p<0.01). Two of these six rats showed histological evidence of precancerous lesions in the mucosa of the colon. This study suggests that the decrease in plasma zinc, tissue zinc and activity of CuZnSOD is associated with development of preneoplastic lesions in the colonic mucosa.     

Anticancer properties of panduratin A isolated from <Emphasis Type="Italic">Boesenbergia pandurata</Emphasis> (Zingiberaceae)

Extract of Boesenbergia pandurata (Kaempferia pandurata) (Zingiberaceae) has been used as a replacement for K. rotunda, the main ingredient of a popular traditional tonic called “jamu” especially for women in Indonesia. From our previous study, ethanolic extract of B. pandurata showed strong inhibitory effects on the growth of cancer cells, similar to ethanolic extract of Curcuma longa. C. longa and its bioactive compound, curcumin, have shown potential anticancer activity in in vitro and in vivo studies and have undergone clinical trials. Panduratin A, a chalcone derivative isolated from B. pandurata, was found to inhibit the growth of MCF-7 human breast cancer and HT-29 human colon adenocarcinoma cells with an IC₅₀ of 3.75 and 6.56 μg/ml, respectively. Panduratin A arrested cancer cells labelled with Annexin-V and propidium iodide in the G0/G1 phase and induced apoptosis in a dose-dependent manner. In an animal model study, male Sprague–Dawley rats were fed with AIN diet containing ethanolic extracts prepared from the equivalent of 4% by weight of dried rhizomes of B. pandurata and C. longa. Aberrant crypt foci (ACFs) were induced by two subcutaneous doses (15 mg/kg body weight) of azoxymethane (AOM) 1 week apart. The rats were killed 10 weeks later, and the ACFs were assessed in the colon. At the dose given to rats, it appeared that the extracts were not toxic. Total ACFs were slightly reduced by B. pandurata extract compared to control group but not significantly different. Extract of B. pandurata may have a protective effect against colon cancer but additional studies using different models, such as a breast cancer model, need to be carried out.     

Early Alterations of Apoptosis and Cell Proliferation in Azoxymethane-initiated Rat Colonic Epithelium

Alterations of apoptosis and cell proliferation in the colonic epithelium of rats after exposure to azoxymethane (AOM) were estimated by means of the terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling (TUNEL) method, measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation, immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and counting of raitotic cells. F344 male rats were given a single s.c. injection of AOM (15 mg/kg body weight) at 6 week of age, and killed 4 h, 8 h, 3 days, and 7 days after the AOM treatment. At 4 h after the treatment, many damaged cells were already observed in the colonic epithelium, and they were positive by TUNEL staining. At 8 h, the number of TUNEL-positive cells was largest. The reduction of DNA synthesis in the colonic epithelium, confirmed by BrdU incorporation, was not distinct in comparison with the mitotic inhibition. There was no remarkable change in PCNA labeling index, except that strong expression of PCNA was detected in many damaged cells. On the 3rd day, the appearance of cell death became infrequent and an increase of cell proliferation occurred. On the 7th day, the expression of TUNEL and the cell proliferation biomarkers were at almost normal levels. These findings suggest that AOM induces apoptosis, which is associated with synchronous inhibition of mitosis. The data also indicate that PCNA immunostaining does not reflect the true proliferation state in the early phase after AOM exposure, probably due to the occurrence of cell cycle arrest or DNA repair.     

Triamterene Suppresses Bombesin-enhanced Peritoneal Metastasis of Intestinal Adenocarcinomas Induced by Azoxymethane

The effects of combined administration of bombesin and the diuretic triamterene on the incidence of peritoneal metastasis of intestinal cancers induced by azoxymethane (AOM) and the labeling index of intestinal cancers were investigated in male inbred Wistar rats. From the start of the experiment, rats were given weekly s.c. injections of AOM (7.4 mg/kg body weight) for 10 weeks and s.c. injections of bombesin (40 μg/kg body weight) every other day, and from week 16, s.c. injections of triamterene (10 and 20 mg/kg body weight) every other day until the end of the experiment in week 45. Bombesin significantly increased the incidence of intestinal tumors and cancer metastasis to the peritoneum in week 45. It also significantly increased the labeling index of intestinal cancers. Although administration of both doses of triamterene with bombesin had little or no influence on the enhancement of intestinal carcinogenesis by bombesin, or the location, histologic type, depth of invasion, or labeling index of intestinal cancers, it significantly reduced the incidence of cancer metastasis. These findings indicate that triamterene suppresses cancer metastasis through a mechanism that does not affect the proliferation of intestinal cancers.     

Regressive Effects of Various Chemopreventive Agents on Azoxymethane-induced Aberrant Crypt Foci in the Rat Colon

Regressive effects of four chemopreventive agents [5-hydroxy-4-(2-phenyl-( E )-ethenyl)-2(5 H fura-none (KYN-54), S-methyl metbanethiosulfonate (MMTS), chlorogenic acid (CA), and piroxicam] on azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon of male F344 rats were examined by dietary exposure. At six weeks of age, 60 rats of groups 1 through 5 received subcutaneous injections of AOM (15 mg/kg body weight) once a week for three weeks. Twelve weeks after the first carcinogen injection, wben the occurrence of ACF was maximal, the rats in groups 2 through 5 were started on diet containing the test chemicals as follows: group 2, KYN-54 (0.02%); group 3, MMTS (0.01%); group 4, CA (0.025%); and group 5, piroxicam (0.0125%). Group 1 (20 rats) was kept on the basal diet alone, and group 6 (12 rate) served as an untreated control. Rats in each group were killed at 6, 12, 18, or 24 weeks after the start of the experiment, and the yield of ACF in the colon of each group at 18 or 24 weeks was compared with that at 12 weeks. The number of ACF per rat colon of each group at 18 or 24 weeks was smaller than that at 12 weeks. The reduction rates at 18 weeks were 7% in group 1 (AOM alone), 11% in group 2 (AOM+KYN-54), 10% in group 3 (AOM+MMTS), 51% in group 4 (AOM + CA) ( P 0.01), and 33% in group 5 (AOM+piroxicam) ( P <0.02), while at 24 weeks they were 12%, 26%, 51% ( P <0.002), 43% ( P <0.05), and 70% ( P <0.001), respectively. These results indicate that chemopreventive agents for large bowel carcinogenesis, i.e., KYN-54, MMTS, CA, and piroxicam, are not only able to prevent the development of ACF, but also can regress ACF, which are regarded as precursor lesions of colorectal cancer.     

Inhibition of Azoxymethane-induced Colon Carcinogenesis in Rat by Green Tea Polyphenol Fraction

The effect of green tea polyphenol fraction (GTP) on azoxymethane(AOM)-induced colon carcino-genesis was investigated in male Fischer rate. The rats were given AOM (7.4 mg/kg body weight) s.c. once a week for 10 weeks. A week after the treatment, they were divided into three groups: AOM-control (26 rats), AOM-GTP1 (26 rats) and AOM-GTP2 (25 rats). AOM-GTPl and AOM-GTP2 groups respectively received 0.01 and 0.1% GTP in drinking water from week 11 to 26. AOM-control group received tap water throughout this experiment. Autopsy on week 26 showed that tumor incidence and average numbers of tumors per rat in the AOM-GTPl and AOM-GTP2 groups were significantly lower than those of the AOM-control group: 38.1% and 47.6% versus 77.3%; 0.6 and 0.7 versus 1.5. Thus, it was concluded that GTP inhibited the development of AOM-indnced colon carcinogenesis. The inhibition by GTP did not show significant dose dependence.     

芥菜籽预防化学诱导小鼠大肠肿瘤的实验研究

目的 评价芥菜籽（MS）对氧化偶氮甲烷（AOM）诱导的小鼠大肠肿瘤的预防作用及其机制。方法 选择60只昆明种小鼠,随机均分为AOM 模型组、AOM+5％MS干预组、AOM+10％MS干预组和正常对照组（生理盐水）。记录各组小鼠有无肿瘤发生及发生数目、大小和位置, 计算平均肿瘤数和肿瘤发生率;HE染色确定肿瘤的组织学类型;免疫组化染色检测肿瘤组织中PCNA蛋白的表达,计算增殖指数（PI）;TUNEL染色检测肿瘤组织凋亡情况,计算凋亡指数（AI）。结果 正常对照组小鼠无肿瘤发生,AOM模型组、5％MS干预组和10％MS干预组小鼠肿瘤发生率分别为86.7％、 60.0％、41.7％,组间差异有统计学意义（P=0.048）; 5％MS干预组平均肿瘤数为1.07±1.10,10％MS干预组为0.67±0.89,均较AOM模型组的2.20±1.21少（P＜0.05）;10％MS干预组PI为32.0±3.9,均低于AOM模型组和5％MS干预组的59.9±4.4和41.7±4.9（P＜0.05）;10％MS干预组AI为15.0±2.4,均高于AOM模型组和5％MS干预组的6.9±1.4和9.3±1.5（P＜0.05）。结论 MS对AOM诱导的小鼠大肠肿瘤具有化学预防作用,其机制为抑制肿瘤细胞增殖和诱导肿瘤细胞凋亡。     

硒对氧化偶氮甲烷所致结肠癌模型大鼠睾丸精原细胞P16蛋白表达的影响

目的观察硒对氧化偶氮甲烷（AOM）所致结肠癌大鼠睾丸精原细胞P16蛋白表达的影响.方法随机将20只3周龄断乳雄性SD大鼠分为：正常对照组、实验对照组、AOM致癌前喂食亚硒酸钠水组、AOM致癌后喂食亚硒酸钠水组.每周腹腔注射AOM 15 mg/kg,持续2周,造成大鼠结肠癌模型.用4 mg/L Na2SeO3水溶液分别在AOM致癌前、后开始进行干预,并持续至实验结果.各组均于34周后取两侧睾丸,用免疫组织化学方法,观察大鼠睾丸精原细胞内P16蛋白的表达,并进行图像分析.结果正常组大鼠的睾丸内未见精原细胞表达P16蛋白,而实验对照组、硒干预AOM致癌组（AOM致癌前、后喂食亚硒酸钠水）的精原细胞P16均呈阳性表达,阳性产物呈深棕色,定位于细胞核中,细胞质内仅有少量表达.实验对照组、AOM致癌前、后喂食亚硒酸钠水组阳性表达逐渐增强,组间具有显著性差异（P〈0.05）.结论硒可增强AOM所致结肠癌大鼠精原细胞P16蛋白的表达.     

Enhanced Formation of Azoxymethane-induced Colorectal Adenocarcinoma in γδ T Lymphocyte-deficient Mice

T cell receptor (TCR) γδ -positive T lymphocytes, which are localized mostly within the intraepithe-lial space of intestinal epithelium, have been suggested to play a role in maintaining the normal configuration of intestinal epithelium. However, the role of TCRγδ -positive T lymphocytes in the formation and progression of colorectal adenocarcinoma that originates from colorectal epithelial cells remains to be elucidated. In this study, TCRαβ and TCRγδ -positive T lymphocyte-deficient mice (homozygous TCRα and TCRδ-gene knockout mice) and the background wild-type mice were administered azoxymethane, and the formation of macroscopic tumors and microscopic aberrant crypt foci in colorectal mucosa were compared among the three types of mice. Well-differentiated adenocarcinoma appeared 5 months after 5 administrations of azoxymethane (10 mg/kg weight) only in a few TCRδ-gene knockout mice and the frequency of the carcinoma-bearing mice was increased at 7 and 9 months after the administration. Aberrant crypt foci were also detected in the colorectal mucosa of TCRδ-gene knockout mice to a greater extent than in colorectal mucosa of TCRδ-gene knockout mice 1 month after the azoxymethane administration. These results suggest that TCRγδ -positive T lymphocytes, which are present mainly in the intraepithelial space, play a role in suppression of the formation and progression of colorectal adenocarcinoma in mice.