Binding Sites of Droloxifene in the Cytosol of 7,12-Dimethylbenz[α]anthracene-induced Rat Mammary Tumor Cells

The binding sites, other than the estrogen receptor (ER), of the antiestrogens droloxifene (DROL, (E)-α-[ p [2-(dimethylamino)ethoxy]-phenyl]-α'-ethyl-3-stilbenol) and tamoxifen (TAM), and estradiol-17β (E₂) in the cytosol of 7,12-dimethylbenz[α]anthracene-induced rat mammary ER-positive tumor cells were studied using a high-performance liquid chromatography (HPLC) gel filtration assay. The cytosol was incubated with ³H-labeled drug with or without unlabeled drug, and separated by HPLC gel filtration. ³H-E₂ produced two major peaks of radioactivity at fractions No. 40 and No. 70. The peak at fraction No. 70 was identified as the ER in an ER-enzyme-immuno assay. This peak was dose-dependently inhibited by unlabeled DROL or TAM, DROL being a more potent inhibitor than TAM. The peak at fraction No. 40 was also inhibited by co-incubation with unlabeled DROL or TAM. ³H-DROL or ³H-TAM provided only one peak at fraction No. 43. This peak was thought to be an antiestrogen binding site (AEBS), because it was inhibited by unlabeled antiestrogen hut not by E₂. The results suggest that the antiestrogens DROL and TAM have a higher affinity for the AEBS than for the ER in the absence of E₂, while in the presence of E₂ both have an affinity for the ER and inhibit E₂ binding to the ER.     

Wakame seaweed suppresses the proliferation of 7,12-dimethylbenz(a)-anthracene-induced mammary tumors in rats.

We examined the anti-tumor proliferation effects of wakame seaweed on 7,12-dimethylbenz(a)-anthracene (DMBA)-induced rat mammary tumor. DMBA was administered to 8-week-old female Sprague-Dawley rats, and rats which developed mammary tumors were assigned randomly to three groups. Commercial rat feed was used in a control group (group I-A), and two feed mixtures were prepared, which contained commercial rat feed blended with wakame at 1.0% (group I-B) and 5.0% (group I-C) by weight. The respective feeds were given to each group for 8 weeks, and changes in mammary tumor size were compared. At the end of the experiment, mammary tumors and thyroid glands were resected to compare their weights. Serum total iodine and thyroxin (T4) levels were measured. Immunohistochemical studies for bromodeoxyuridine (BrdU) labeling, transforming growth factor (TGF)-beta, and apoptosis were carried out in the resected tumor. Significant suppression of tumor growth was observed in groups I-B and I-C compared with I-A. In groups I-B and I-C, the weights of resected mammary tumors were significantly lower and serum total iodine concentration was significantly higher than in I-A. BrdU indices were significantly lower in groups I-B and I-C, compared with I-A. TGF-beta and apoptotic index were inversely related to BrdU. These results suggest that iodine is transported from the serum into mammary tissues and induces apoptosis through the expression of TGF-beta. In conclusion, wakame suppressed the proliferation of DMBA-induced mammary tumors.     

Effects of dehydroepiandrosterone and other sex steroid hormones on mammary carcinogenesis by direct injection of 7, 12-dimethylbenz(a) anthracene (DMBA) in hyperprolactinemic female rats

The present study was performed to investigate theeffects of dehydroepiandrosterone (DHEA) compared with those ofsex steroid hormones on the mammary tumor inducedby local injection of 7, 12-dimethylbenz(a)anthracene (DMBA) inhyperprolactinemic female rats. Under sustained hyperprolactinemia induced bypimozide (PMZ) from day 21, DMBA was injectedlocally into the mammary glandular tissues on day73. Rats were divided into 5 groups asfollows; steroid free (DP group), 17 -estradiol (DP+ E2 group), testosterone (DP + T group),progesterone (DP + Prog group), or dehydroepiandrosterone (DP+ DHA group). The growth pattern and histologicalclassification of the tumor in these 5 groupsand rats treated only with DMBA (D group)were examined. All of the tumors grew toa size of 10 mm in diameter andafter retaining the size for a certain duration,increased the size rapidly again (onset of rapidtumor growth). The period from the day ofDMBA administration to that of onset of therapid tumor growth in DP group was shorterthan in D group, and the period inDP + DHA was longer than DP groupand longest in steroid-treated groups. The incidence ofadenocarcinoma was 2 tumors/16 animals in D group,9/11 in DP group, 5/11 in DP +Prog group, 2/7 in DP + E2 group,2/8 in DP + T group, and 0/10in DP + DHA group. The incidence ofadenocarcinoma in each steroid group except in DP+ Prog group was lower than in DPgroup. These results suggest that prolactin (PRL) increasesthe incidence of adenocarcinoma in the DMBA-induced mammarytumor model, and DHEA especially decreases the incidenceof adenocarcinoma.     

The Effect of Castration on the Synthesis and Secretion of Proteins in 7,12-Dimethylbenz(a)anthracene-Induced Rat Mammary Tumors

The effect of castration on the incorporation of p⁵S]methionine into secreted proteins in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, was investigated. Biopsy specimens were obtained from 19 tumors, 0, 24, 48, 72, and 96 h after castration. In 14 tumors, castration induced an increase in the incorporation (mean of 5-fold), reaching the maximal level after 24 h (3 tumors), 48 h (7 tumors), 72 h (3 tumors), and after 96 h (1 tumor). In three tumors castration did not alter the incorporation rate, while in two tumors incorporation declined immediately after castration. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the labeled secreted protein showed that castration did not decrease or increase significantly the incorporation of [³⁵S]methionine into any of the major labeled proteins. Conclusion: tumor regression following hormonal deprivation is apparently preceded by an increased synthesis of secreted proteins. However, no qualitative differences in any major labeled proteins could be observed.     

Correlation of Tumour Response with Starting Tumour Size and Dose of Tamoxifen in an N-Methyl-N-Nitrosourea (NMU)-Induced Rat Mammary Cancer Model

Background: The aim of this preliminary study was to address variations of responses observed with different starting tumor sizes of 10 and 15 mm, and the effects of different doses of tamoxifen (TAM) on experimental rat mammary tumors. Materials and Methods: Thirty-five inbred female Sprague Dawley rats aged 43 days were administered with three weekly doses of N-methyl-N-nitrosourea (NMU) intraperitoneally (ip) at 50 mg/kg body weight. Animals were randomized (beginning from 10 mm tumor size) into four TAM-treated (50, 100, 200 and 500 μg/day) groups of six animals each, and another group (n=6) treated with TAM 100 μg/day at starting tumour size of 15 mm. The animals were treated by oral gavage daily for 8 weeks before sacrifice. Results: Serum urea and creatinine, and overall physical tumor burden were significantly modulated in animals treated with variable doses of TAM compared to the untreated controls (n=5). Final body weight and tumor number were significantly different in the 10 mm-treated animals compared to those treated at 15 mm. There were no significant differences in histopathological features among all the groups. Conclusions: Our findings suggest the importance of standardizing tumour size and drug doses before initiation of treatment, particularly in the direct comparison of basic end-tumour physical parameters.     

Effects of Reproduction on Spontaneous Development of Endometrial Adenocarcinomas and Mammary Tumors in Donryu Rats

Effects of reproduction on spontaneous development of uterine endometrial adenocarcinomas and mammary tumors in Donryu rats were investigated. While the incidence of endometrial adenocarcinomas in Donryu rats was not influenced by a single reproductive experience (SRE), it showed a tendency to decrease in animals having three reproductive experiences (TRE), compared to the nulliparous case (NRE). In addition, both SRE and TRE animals showed delayed occurrence and decreased incidences and mean numbers of mammary tumors, along with reduced incidences of proliferative lesions in the pituitary gland and mucinous epithelium in the vagina. The appearancetime and incidences of persistent estrus in TRE rats were delayed and low, respectively, compared to the SRE and NRE values. The hormonal environment was altered in both groups, the prolactin level in TRE especially being decreased. These results suggest that suppression of the occurrence of endometrial adenocarcinomas and mammary tumors in rats experiencing reproduction is associated with change in the hormonal milieu.     

Effects of sequential and combined endocrine therapies on the growth of 7,12-dimethylbenz [alpha] anthracene-induced rat mammary carcinoma

Sixty-six female Sprague-Dawley (SD) rats with 7,12-dimethylbenz [alpha] anthracene(DMBA)-induced rat mammary cancer were divided into five groups: tamoxifen (TAM), medroxyprogesterone acetate (MPA), TAM + MPA, ovariectomy (Ovex), control (no treatment). An antitumor effect was shown in each treated group. Thirty-six (72%) out of 50 treated animals responded to the first endocrine therapy. Moreover, tumors disappeared completely from 21 out of the 36 animals, and no new tumors were seen until the 12th week. The facts suggest experimental hormone, when compared to clinical, therapy in DMBA-induced tumors to have a higher response rate. A total of 14 out of 50 tumors failed to respond to the first treatment (resistant tumor). There was no significant difference in the proportion of resistant tumors among the four treated groups. When other endocrine therapies were tried out of resistant tumors, seven out of the 14 responded, the resistant tumors in the TAM group responding significantly well to the other endocrine therapies compared to those in the MPA group (P less than 0.05). These results suggest the possibility of resistant tumors responding to different types of endocrine therapy.     

Inhibition of Mammary Tumors by Pretreatment with 17β-Estradiol in F344 Rats Induced with N-Methyl-N-nitrosourea

The influence of 17β-estradiol (E2) and prolactin was studied on N-methyl-N-nitrosourea (MNU)-induced mammary carcinomas (MCAs) in rats. MNU was intravenously injected once into seven-week-old female F344 rats at a dose of 50 mg/kg body weight. Groups of rats also received either 2.5 mg of E2 or a continuous supply of prolactin and/or growth hormone via transplanted MtT/F84 (mammo-somatotropic pituitary tumor). Rats were observed for up to 36 weeks after MNU administration. Although simultaneous administration of MNU and E2 did not much affect the occurrence of MCAs as compared to administration of MNU alone, rats treated with 2.5 mg of E2 for two weeks before MNU administration had significantly reduced occurrence of MCAs compared to those given MNU alone. In contrast, rats with MNU plus MtT/F84 showed high incidence and shortened latency of MCAs and they also had a high incidence of clitorial gland hyperplasias. Average pituitary weights and serum prolactin levels in E2-treated rats were greatly increased compared to those of MNU-alone rats. Average serum E2 levels were about 100 ng/ml in E2-treated rats and 0.05 ng/ml in rats without E2 treatment. Serum prolactin levels were greatly increased in rats with MtT/F84. The results indicated that pretreatment with E2 before MNU administration was inhibitory while increased prolactin caused by grafting MtT/F84 after MNU injection was promotive for the occurrence of MCAs in female F344 rats.     

Plantago major Protective Effects on Antioxidant Status after Administration of 7,12-Dimethylbenz(a)anthracene in Rats

Aim: The present study was designed to evaluate effects of Plantago major extract on oxidative status in Wistaralbino rats administrated 7,12-dimethylbenz(a)anthracene (DMBA). Methods: Rats were divided into three equalgroups of 6 animals each: Group 1 controls, group 2 treated with DMBA (100 mg/kg, single dose) and group 3receiving the DMBA and the aqueous extract at 100 mg/kg/d for 60 days. Results: Significant decrease in catalase(P<0.05), carbonic anhydrase (p≤0.01), reduced glutathione (GSH) (P<0.01) and total protein (P<0.01) valueswas observed in the DMBA group compared with the healthy controls and DMBA + Plantago major groups.Conclusion: The results suggest preventive effects of Plantago major on DMBA induced oxidative damage inWistar albino rats that might be due to decreased free radical generation.     

Secreted Frizzled-related Protein 2 (SFRP2) is Highly Expressed in Canine Mammary Gland Tumors but not in Normal Mammary Glands

Canine mammary gland tumor (MGT) is the commonest tumor in female dogs and a good animal model of human breast cancer. A group of newly identified genes encoding secreted frizzled-related proteins (SFRP) have been implicated in apoptosis regulation and tumorigenesis. Canine mammary tissues from 50 spontaneous MGTs and 10 normal mammary glands (MGs) were obtained from surgically excised specimens and analyzed for expression of SFRP2, beta-catenin, and cyclin D1. By RT-PCR and in situ hybridization, SFRP2 gene was found abundantly expressed in neoplastic mammary tissues but not in normal mammary tissues, suggesting that SFRP2 may contribute as a tumor marker in canine MGTs. By immunohistochemical staining, the immunoreactivity of the SFRP2 protein was detected in more diverse areas than SFRP2 mRNA expression, including nuclei or/and cytoplasm and extracellular matrix of the tumor. In tumor masses, beta-catenin lost its tight association with the membrane and diffused into the nucleus. The expression of beta-catenin (79.4% positive) and cyclin D1 (71.4% positive) was also increased in MGTs. In the course of tumor progression, SFRP2 mRNA ( p < 0.05) and beta-catenin protein ( p < 0.01) steadily increased but not in cyclin D1. The level of SFRP2 was linearly correlated with its downstream target beta-catenin ( p < 0.05), but not correlated with cyclin D1 ( p < 0.5). As revealed in this study, the exclusive overexpression of SFRP2 in canine MGTs suggests that SFRP2 is a potential candidate gene for further investigation of mammary tumorigenesis and complex etiology of the canine model of mammary neoplasms.     

Long-term Exposure to the Anti-inflammatory Agent Phenylbutazone Induces Kidney Tumors in Rats and Liver Tumors in Mice

Long-term toxicity and carcinogenicity of phenylbutazone, a nonsteroidal anti-inflammatory drug, were evaluated in F344/N rats and B6C3F1 mice. In 2-year studies, phenylbutazone was given in corn oil by gavage 5 days per week to groups of 50 rats of each sex at doses of 0, 50, or 100 mg/kg body weight, and to groups of 50 mice at doses of 0, 150, or 300 mg/kg body weight. Body weights and survival were similar among groups. Major target organs are kidneys in rats, and liver in mice. Kidney: inflammation, papillary necrosis, and mineralization in both sexes of rats, and hyperplasia and dilatation of the pelvis epithelium, and cysts in female rats. Uncommon tubular cell tumors of the kidney were found in 13 exposed rats: 5 in the 50 mg group and 4 in the 100 mg group of males; 4 in dosed female rats; none in controls. In female rats, dose-related increases in hyperplasia of the pelvis transitional epithelium, and 2 carcinomas were discovered. Urinary bladder: papillomas of the transitional epithelium were seen in 2 low-dose male and in 1 low-dose female rats. Forestomach: ulcers in rats, with acanthosis, hyperkeratosis, and basal cell hyperplasia in female rats; however, no neoplasms were associated with these lesions. Liver: primarily in male mice exposed to. phenylbutazone, hemorrhage, centrilobular cytomegaly and karyomegaly, fatty metamorphosis, cellular degeneration, and coagulative necrosis were seen; clear cell foci were observed in male mice. In summary, under the conditions of these 2-year oral intubation studies, phenylbutazone is associated with renal carcinogenicity in rats, as evidenced by increases in tubular cell neoplasms in both sexes. Evidence of carcinogenicity for male mice was shown by increased incidences and multiplicity of liver tumors. No carcinogenic activity was found for female mice.     

叶绿酸对7，2—二甲基苯蒽诱发大鼠乳腺癌的影响

本文研究了叶绿酸（ＣＨＬ）对７，１２—二甲基苯蒽（ＤＭＢＡ）诱发大鼠乳腺癌的影响。４３天龄雌性Ｓｐｒａｇｕｅ—Ｄａｗｌｅｙ大鼠随机分为３组，每组２５只。给ＤＭＢＡ前Ⅰ组给自来水，Ⅱ、Ⅲ组在饮水中加ＣＨＬ１．５ｍｍｏｌ，ｌｗｋ后每只大鼠一次灌胃给ＤＭＢＡ１０ｍｇ．给ＤＭＢＡ后Ⅰ、Ⅱ组给自来水，Ⅲ组饮水中继续加ＣＨＬ，持续２４ｗｋ。结果３组大鼠乳癌发生率分别为５０．０％、３３．３％和２３．８％．给ＣＨＬ的两组与对照组比较有下降趋势，但差异无显著性，说明ＣＨＬ的抗癌作用还有待进一步证实。实验中未发现ＣＨＬ有促癌作用。     

Estrogen Disrupts Chemokine-Mediated Chemokine Release from Mammary Cells: Implications for the Interplay between Estrogen and IP-10 in the Regulation of Mammary Tumor Formation

Chemokines are pro-inflammatory cytokines that function to attract immune cells to the sites of tissue inflammation, injury or infection. We have formulated the hypothesis that release of one chemokine can serve, in a local paracrine or endocrine fashion, to induce the release of other chemokines from neighboring mammary cells. We set out to investigate whether specific chemokines could promote the release of other chemokine members from mammary cells, and whether estrogen could serve to disrupt the release of these chemokines from mammary cells. We found that treatment with the chemokine IP-10 resulted in significant increases in the amount of MIP-1alpha and MCP-1/JE released from murine mammary cells. Estrogen co-treatment significantly blocked the ability of IP-10 to trigger the release of MIP-1alpha and MCP-1/JE. Suppressive effects of estrogen were reversed upon co-treatment with 4-hydroxytamoxifen. Estrogen treatment significantly decreased expression of proteins corresponding to the chemokine receptors CXCR3 and CCR5 on mammary cells. Exposure of female mice to IP-10 in vivo significantly decreased the ability of estrogen to support the growth of CCL-51-based tumors in mammary tissue. Our results suggest that exposure of mammary tissue to estrogen may decrease the release of local chemokines from mammary cells, potentially increasing the risk of tumor growth through decreased immune surveillance. Ongoing studies are investigating the possible mechanisms through which IP-10 stimulates the release of chemokines from mammary cells, and how the action of IP-10 may serve to decrease mammary tumor formation.     

Optimization of 7,12-dimethylbenz(a)anthracene-induced rat epithelial ovarian tumors

Ovarian carcinoma is the second most common malignant tumor of the female reproductive system and an notable cause of cancer death. The detection and diagnosis of early ovarian carcinomas are still clinical challenges, which calls for imaging studies using early ovarian carcinoma animal models. The present study aimed to optimize the 7,12-dimethylbenz(a)anthracene (DMBA)-induced model of rat ovarian tumors by investigating the delivery methods, induction dose and time of DMBA exposure, and explored the morphological features of tumors using MRI. Three schemes were performed. In scheme one the ovary was covered with absorbable hemostatic gauze loaded with a high concentration of liquid DMBA. For this scheme, 150 Sprague-Dawley rats were divided into three groups depending on the DMBA dose (1.0, 2.0 and 3.0 mg). In scheme two DMBA solution was injected under the ovarian capsule. For this scheme, 159 rats were divided into 0.5, 1.0 and 1.5 mg DMBA groups. In scheme three the ovary was covered with absorbable gauze loaded with a high concentration of solid DMBA. For this scheme 161 rats were divided into 1.0, 2.0 and 3.0 mg DMBA groups. Each group of the three schemes was further subdivided into 60-, 90-, 120-, 150- and 180-day groups. In scheme two, the tumor formation rate was 75.6% (99/131), which was the highest in the 1.5 mg group (86.4%, 38/44) and reached 100% (10/10) on day 120. The induced tumors were serous in 93.9% (93/99) of tumors. Borderline ovarian tumors accounted for 19.2% (19/99) of all tumors, and ovarian cancer accounted for 46.5% (46/99). The mean maximum diameter (MMD) of borderline ovarian tumors was 10.29±3.41 mm, and that of ovarian cancer was 15.19±7.10 mm. MMD of the solid components increased with increasing malignancy. Cystic, cystic-solid and solid tumors were observed. The ovarian subcapsular injection of 1.5 mg DMBA was the best scheme for the rat ovarian tumor model. The present model is ideal for investigating the occurrence, development and imaging of ovarian tumors.     

Influence of Esculetin on Incidence, Proliferation, and Cell Kinetics of Mammary Carcinomas Induced by 7,12-Dimethylbenz[α]anthracene in Rats on High- and Low-fat Diets

The effects of a high-fat diet and esculetin were investigated on 7,12-dimethylbenz[α]anthracene (DMBA)-induced mammary carcinogenesis in female Sprague-Dawley rats. Rats were given a 5-mg dose of DMBA. Seven days later, they were fed either a high-fat (20% soybean oil) or low-fat (0.5% soybean oil) diet. A half of the rats received diets containing 0.03% esculetin. Esculetin significantly inhibited tumor incidence, growth and cell kinetics of the tumor in the rats fed the high-fat and the low-fat diets. Our findings indicate that DMBA-induced mammary tumorigenesis is affected by lipoxygenase products.