Anthracene-induced Rat Mammary Tumors Research Articles

A high-corn-oil diet strongly stimulates mammary carcinogenesis, while a high-extra-virgin-olive-oil diet has a weak effect, through changes in metabolism, immune system function and proliferation/apoptosis pathways

Breast cancer is the most common malignancy in women worldwide, and dietary lipids are important environmental factors influencing its etiology. We have investigated the effects, and the mechanisms associated, of high-fat diets on 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. Animals were fed a low-fat, a high-corn-oil (HCO) or a high-extra-virgin-olive-oil (HOO) diet from weaning or after induction. The HCO diet had a clear stimulating effect on mammary carcinogenesis, especially when dietary intervention started after induction, whereas the tumors from HOO diet groups exhibited clinical and morphological characteristics similar to those from low-fat controls. Transcriptomic and further protein and immunohistochemical analyses of tumors also indicated different modulatory effects of high-fat diets affecting relevant biological functions: metabolism, immunosurveillance and proliferation/apoptosis pathways. Thus, the results suggested different metabolic adaptations with increased glycolysis by effect of HOO diet. Moreover, leukocyte tumor infiltration and inflammation mediators showed increased cytotoxic T cells and decreased TGFβ1 expression by the HOO diet, while the HCO one increased arginase expression and IL-1α plasma levels. Furthermore, the study of proteins controlling proliferation/apoptosis pathways (Sema3A, Stat5, Smad1, Casp3) suggested an increase in proliferation by the HCO diet and an increase of apoptosis by the diet rich in olive oil. In conclusion, the HCO diet clearly stimulated mammary carcinogenesis, especially in the promotion phase, and induced molecular changes suggesting increased tumor proliferation/apoptosis balance and a proinflammatory microenvironment. The HOO diet, despite being high fat, had a weaker effect on tumorigenesis probably related to metabolic adaptations, enhanced immunosurveillance and increased apoptosis.

In Vivo Assessment of Anti-Tumor Activity of Methyl 2'-Methyl-1,3-dioxo-1,1',2',3,5',6',7',7a'-octahydrospiro[indene-2,3'-pyrrolizidine]-2' carboxylate (6) on 7,12 dimethylbenz(a) Anthracene-Induced Mammary Tumors in Rat

Objective- Breast cancer is one of the most serious problems in oncology. Alkaloids pyrrolizidine are widely found in nature and exhibited versatile biological activities. The aim of the present study was to assess anti-tumor activity of methyl 1,3-dioxo-1,1',2',3,5',6',7',7a'-octahydrospiro[indene-2,3'-pyrrolizidine]-2'-carboxylate (6) on 7,12 dimethylbenz(a) anthracene (DMBA)-induced mammary tumors in rat. Design- Experimental study. Animals- Twenty-one female Wistar ats Procedures- 7-Week-old female Wistar rats (180 g body weight) were randomized into three groups of seven animals each. DMBA-induced mammary tumors was achived using DMBA dissolved in 1ml of vehicle (0.5 ml of DMSO plus 0.5 ml of saline) and injected by subcutaneous injection beneath the mammary gland on either side. Tumor yield and size were stabilized after 90 days with the initiation of DMBA. Healthy intact animals (NC) were considered as a normal control feeding on pellets and tap water. In group DMBA, the tumor was induced by DMBA. In group DMBA/7d the animals with tumor received 100 µL 6 dissolved in DMSO (0.25 µM) solution intraperitoneally for one week. Synthesis of 6 was accomplished and the structure of new product was assigned by their 1H/13C NMR, IR, CH-COSY and mass spectral data as well as the elemental analysis. Results- The overall tumor analysis showed that 6 treatment significantly inhibited the breast tumor incidence, tumor multiplicity, and tumor size in the DMBA-initiated rat model. 6 treatment significantly inhibited the total volume of tumors per rat. Conclusion and Clinical Relevance-The findings of the present study showed that the 6 treatment was significantly effective in reduction of tumors versus the cancer controls.

Whey Protein Hydrolysate but not Whole Whey Protein Protects Against 7,12-Dimethylbenz(a)anthracene-Induced Mammary Tumors in Rats

Effects of intact and processed bovine milk proteins on development of chemically induced mammary tumors in female rats were compared. AIN-93G diets were made with 20% casein (CAS), casein hydrolysate (CASH), intact whey protein (IWP), or whey protein hydrolysate (WPH). Pregnant Sprague-Dawley rats were fed the diets starting at Gestational Day 4. Offspring were fed the same diet. At 50 days, female offspring (44–49/group) were gavaged with sesame oil containing 80 mg/kg of the mammary carcinogen dimethylbenzanthracene (DMBA) and euthanized 62 days posttreatment. Rats fed WPH had an adenocarcinoma incidence of 17% compared to the rats fed CAS, CASH, and IWP diets (34%, 33%, and 36% respectively) (P < 0.001). Median palpable tumor latency for rats fed WPH was greater (61 days, P < 0.001) compared to CAS (44 days), CASH (42 days) and IWP (45 days). Tumor multiplicity was also lower (1.5 vs. 3.0, P < 0.05) in rats fed WPH than in CAS and CASH fed groups. Results demonstrate that hydrolytic processing of whey protein is required for this diet to be effective in reducing DMBA-induced mammary tumors. The bioactive compounds produced during whey protein processing and mechanisms underlying the anticancer effects of WPH are yet to be identified.

Abstract 4528: Genotoxic and estrogenic properties of new antiestrogens

Abstract Long-term administration of tamoxifen (TAM) to breast cancer patients and women at high risk of developing this disease gives serious side effects, including endometrial cancer. These side-effects are due to the DNA damaging and/or estrogenic action of the drug. Several antiestrogens have been developed in last three decades, but were dropped out from the clinical trials due to their undesirable effects on the uterus. To avoid this serious side-effect, we have synthesized a new triphenylethylene antiestrogen, E-3-{4-[(E)-4-chloro-1-(4-hydroxyphenyl)-2-phenylbut-1-enyl]-phenyl} acrylic acid (SS1020) and a new benzopyran antiestrogen, 2E-3-{4-[(7-hydroxy-2-oxo-3-phenyl-2H-chromen-4-yl)-methyl]-phenyl}-acrylic acid (SS5020). SS1020 and SS5020 had antitumor potential much higher than that of TAM or other antiestrogens at the human equivalent doses (TAM 0.3-1.0 mg/kg/day), against 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats or human MCF-7 breast cancer xenograft implanted into athymic nude mice. To evaluate their genotoxic activity, the capability of forming hepatic DNA adducts in rats was determined. When rats were treated orally at a dose molar equivalent to TAM (20 mg/kg/day), a high level (∼1 adduct/105 nucleotides) of DNA adducts was detected in TAM-treated rats whereas no DNA adducts were observed in rats treated with SS1020 or SS5020. To evaluate the estrogenic potential, the uterotrophic activity of our compounds was determined in ovariectomized (OVX) rats at a dose molar equivalent to TAM (10 mg/kg/day), which is 30 times higher than the human equivalent dose of TAM. TAM had high uterotrophic activity while SS1020 and SS5020 did not present significant uterotrophic activity. Therefore, SS1020 and SS5020, lacking estrogenic and genotoxic actions, are expected not to induce reproductive cancers. Interestingly, the antitumor activities of several antiestrogens examined were, rather, increased significantly to the lowering uterotrophic activity evaluated with OVX-rats, but not to their antiproliferative activities against cultured estrogen receptor positive human MCF-7 breast cancer cells. The use of uterotrophic assay, not antiproliferative assay, may be a suitable screening system to exploit antiestrogen alternatives. Non-genotoxic and non-estrogenic SS1020 and SS5020, having strong antitumor potency superior to that of TAM, could be safer alternatives for breast cancer therapy and prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4528. doi:10.1158/1538-7445.AM2011-4528

Glycine soya diet synergistically enhances the suppressive effect of tamoxifen and inhibits tamoxifen-promoted hepatocarcinogenesis in 7,12-dimethylbenz[α]anthracene-induced rat mammary tumor model

There is increasing interest in phytoestrogens as potential alternatives to synthetic selective estrogen receptor modulators (SERMs) in the prevention and therapy of breast cancer. The present study is aimed at determining whether dietary glycine soya (Glycine max seeds; GS), which is rich in phytoestrogens, can enhance the anti breast cancer efficacy of the SERM tamoxifen (TAM) and the effect of TAM and GS, either alone or in combination, on DMBA-initiated hepatocarcinogenesis in rat. For determination of enhancing effect, rats bearing palpable 7, 12-dimethylbenz[α] anthracene (DMBA)-induced mammary tumors were treated with TAM (10mgkg−1/day) while being fed AIN-93G diet with or without added GS (3×104mgkg−1), and the tumor growth was monitored up to 5weeks of treatment. For determining the effect on hepatocarcinogenesis, DMBA-initiated rats were exposed to TAM and dietary GS as above for 6weeks during promotion stage in a medium-term bioassay, and the development of placental form of glutathione-S-transferase (GST-P)-expressing preneoplastic liver lesions was quantified. Exposure to both TAM and dietary GS enhanced the anti tumor efficacy of TAM via a combination of tumor cell apoptosis (determined by TUNEL) and inhibition of tumor cell proliferation (determined by PCNA immunostaining) and suppressed the growth of GST-P-positive liver lesions. The findings show that dietary GS enhances the therapeutic efficacy of TAM against mammary tumors and minimizes TAM’s hepatocarcinogenesis promotion potential.

Consumption of commercial whole and non-fat milk increases the incidence of 7,12-dimethylbenz( a)anthracene-induced mammary tumors in rats

Background: Breast cancer has become the most common cancer among women worldwide. Although the consumptions of milk and dairy products were considered to be a risk factor for breast cancer in some epidemiological studies, the results were inconsistent. Methods: In the present study, female Sprague–Dawley rats received a single oral dose of 5 mg 7,12-dimethylbenz( a)anthracene (DMBA). One week later, the animals were divided into four groups: whole milk (WM), artificial whole milk (A-WM), non-fat milk (NFM) or artificial non-fat milk (A-NFM) mixed with commercial powder chow. Rats were palpated weekly to monitor tumor development. At week 20 after DMBA administration, rats were decapitated and the volume and weight of mammary tumor were recorded. Results: Tumor incidence, the cumulative number of tumors and the sums of tumor volume were higher in the WM and NFM groups than in the A-WM and A-NFM groups both at palpation and at autopsy. Conclusion: Combining our previous studies, we found the consumption of milk promoted the development of DMBA-induced mammary tumors in rats independent of the fat level.

Dipalmitoylation of radicicol results in improved efficacy against tumor growth and angiogenesis in vivo

Tumor-related angiogenesis is likely to be a potential target for the treatment of cancer. One key to develop this angiostatic strategy would be to find useful angiogenesis inhibitors. Here we report the effects of radicicol, a microbial angiogenesis inhibitor that we previously identified using the chorioallantoic membrane assay, and its novel analog, 14,16-dipalmitoyl-radicicol, on tumor angiogenesis and growth. As expected for agents containing a penolic hydroxyl group, systemic administration of radicicol had little or no effect on neovascularization triggered by a M5076 mouse tumor cell line or a RMT-1 rat mammary carcinoma cell line established from autochthonous rat mammary tumors induced by 7,12-dimethylbenz[a]anthracene in a mouse dorsal air sac assay system. The agent did not show growth-inhibitory activity against either transplantable M5076 tumors or autochthonous 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors. In contrast, 14,16-dipalmitoyl-radicicol potently suppressed tumor angiogenesis and growth in these experimental models. Furthermore, the analog significantly prolonged the survival rate of M5076-implanted mice. Although not stronger than radicicol, it dose-dependently inhibited embryonic angiogenesis in the chorioallantoic membrane assay, the dose required for half-maximal inhibition (ID(50)) value being 23 microg (27 nmol) per egg, and showed concentration-dependent antiproliferative activity against microvascular endothelial cells in vitro. These data suggest that 14,16-dipalmitoyl-radicicol is a promising antitumor agent with antiangiogenic activity.

Anti-tumor promoting potential of luteolin against 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats

In this study, we evaluated the anti-tumor potential of luteolin (30mg/kg, p.o.), combined with cyclophosphamide (10mg/kg, i.p.) (LU+CYC) orally administered for 20 days; and CYC individually for 10 days against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in Wistar rats. Combination treatment (LU+CYC) inhibited the incidence rate of tumors and decreased tumor volume significantly without changing the total body weight of the animals. Long-term treatment did not show any apparent toxicity in rats. The CYC-treated group showed potential reduction of tumor volume (74%), severe toxicity, and loss of body weight. In order to elucidate the anticancer mechanism of luteolin, antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) generation in the liver, kidney and breast, as well as protein profiles, were also examined. Biochemical analysis of the combination-treated group showed significant (P<0.01; P<0.05) inhibition of lipid peroxide (LPx) formation (oxygen-free radicals), the level and the activity of SOD, CAT and GPx were found to be very high than the LU and CYC individually treated rats at a 30mg/kg dose. 2D gel electrophoresis analysis revealed that (56kDa) high molecular weight protein was detected in tumors of rats receiving combination treatment than the cancer controls. The biological significance of that protein involved for the dysfunction of cancer cells and induces apoptosis. Histopathological changes also confirmed the formation of tumor tubules and neovascularization after the treatment. Overall, these results suggest that the combination treatment provided antioxidant defense with strong chemopreventive activity against the genesis of DMBA-induced mammary tumors.

In Vitro Actions on Human Cancer Cells and the Liquid Chromatography−Mass Spectrometry/Mass Spectrometry Fingerprint of Phytochemicals in Rice Protein Isolate

Rice protein isolate (RPI) has been reported to reduce the incidence of 7,12-dimethylbenz[a]anthracene-induced mammary tumors in rats. To determine the potential role of phytochemicals associated with the RPI, we studied in vitro antitumor activities of an ether fraction from RPI using human tumor cell lines, including two human breast carcinoma cell lines (MDA-MB-453 and MCF-7) and two myeloma cell lines (RPMI-8226 and IM-9). Concentration-dependent antiproliferative effects of the ether fraction were observed in all cell lines using the standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Fraction-induced apoptosis (P < 0.05) was detected in all cell lines, and this was associated with the induction of proapoptotic bax protein and cdk inhibitors (p21) and the suppression of cdk4 and cyclin D1 activity. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) with both positive and negative modes was used to analyze the phytochemicals in the ether fraction from RPI. Fifty-seven phytochemicals were identified or characterized by their diagnostic fragmentation patterns and direct comparison with the authentic standards on the basis of electrospray ionization-MS/MS data. The major components bound to RPI were lysoglycerophospholipids, fatty acids, and fatty acid 3-[2-(2,3-dihydroxy-propoxycarbonyl)-2-hydroxy-ethoxy]-2-hydroxy-propyl esters.

Increased expression of peripheral benzodiazepine receptor (PBR) in dimethylbenz[a]anthracene-induced mammary tumors in rats

Expression of peripheral benzodiazepine receptors (PBR) has been found in every tissue examined; however, it is most abundant in steroid-producing tissues. Although the primary function of PBR is the regulation of steroidogenesis, its existence in nonsteroidogenic tissues as well as in other cellular compartments including the nucleus suggests that there may be other roles for PBR. Our laboratory reported earlier a significant increase of PBR density in the nucleus of DMBA-induced malignant submandibular glands of rats, suggesting a role of PBR in nuclear events of peripheral tissues. Since then numerous studies have demonstrated the abundance of PBR in tumors. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. Based on studies with a battery of human breast cancer cell lines and several human tissue biopsies, Hardwick et al. suggested that PBR expression, nuclear localization, and PBR-mediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression. The purpose of the present study is to confirm this hypothesis by developing an animal breast cancer model and correlating the above events with the breast cancer. Weanling rats were maintained on a diet containing animal protein (casein) for 30 days and then a single dose of DMBA in sesame oil (80 mg/kg) was administered by gavage to the animals. Control animals received the vehicle only. After 122 days of DMBA administration, the animals were sacrificed. All tumors were detected by palpation. B(max) of PBRs was 52.6% and 128.4% higher in the non-aggressive and aggressive cancer tissues, respectively, than that in normal tissues. Cholesterol uptake into isolated nuclei was found to be higher in both non-aggressive and aggressive tumor breast tissue than that in control tissue. There was also corresponding increase in B(max) of PBRs in the nucleus of cancer tissues. Furthermore, the nuclear nucleoside triphosphatase (NTPase) activity was found to be higher in aggressive tumor tissues than that in non-aggressive tumor tissues. In conclusion, these data suggest that PBR ligand binding, and PBR-mediated cholesterol transport into the nucleus may be involved in the development of mammary gland adenocarcinoma, thus participating in the advancement of the disease.

Inducible nitric oxide synthase expression of tumor and stromal cells is associated with the progression of 7,12-dimethylbenz[ a]anthracene-induced rat mammary tumors

The purpose of this study was to investigate the alteration of inducible nitric oxide synthase (iNOS) expression in rodent mammary tumors. We examined iNOS expression by immunohistochemistry and Western blot analysis in 7,12-dimethylbenz[ a]anthracene (DMBA)-induced rat mammary tumors. In Western blot analysis, invasive carcinomas showed strong expression of iNOS; however, carcinomas in situ, atypical tumors and normal mammary tissue revealed insignificant expression. In immunohistochemistry, tumors revealed positive immunoreactivity in either tumor epithelial, stromal or endothelial cells. In particular, invasive carcinomas showed strong expression at the tumor cells bordering on glandular lumen containing necrotic or apoptotic nuclear debris. Invasive carcinomas showed higher positive immunoreactivity of iNOS compared with normal mammary tissue, atypical tumors, and carcinomas in situ. Stromal iNOS expression was correlated with apoptotic count. These results suggest that iNOS expression of tumor and stromal cells is associated with the progression of DMBA-induced rat mammary tumors.

Steroid-sensitive gene-1 is an androgen-regulated gene expressed in prostatic smooth muscle cells in vivo.

Steroid-sensitive gene-1 (SSG1) is a novel gene we cloned, found regulated by 17beta-estradiol in the rat uterus and mammary gland, and over-expressed in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. We show here that SSG1 mRNA and protein expression are regulated by androgens in the rat ventral prostate. Increases in SSG1 mRNA levels were detected by Northern blotting after 24 h and reached a 27-fold peak 96 h following castration, relative to SSG1 mRNA expression in sham-operated rats. Dihydrotestosterone or testosterone supplementation of castrated rats prevented this rise in SSG1 mRNA. In contrast with SSG1 mRNA expression, SSG1 protein was decreased 16-fold 2 weeks following castration but was at control levels in the prostates of castrated rats receiving dihydrotestosterone or testosterone. Although SSG1 is regulated by androgens in vivo, treatment of LnCap cells with dihydrotestosterone, cyproterone acetate or flutamide did not result in the regulation of SSG1 protein levels in vitro. Immunofluorescence studies show that SSG1 is mainly expressed in prostatic smooth muscle cells. These results indicate that SSG1 is an androgen-regulated gene that is expressed in the smooth muscle component of the rat ventral prostate in vivo.

Bifidobacterium longum as a delivery system for gene therapy of chemically induced rat mammary tumors.

A fundamental obstacle in cancer gene therapy is the specific targeting of therapy directly to a solid tumor, and no systemic delivery system yet exists. A strain of domestic bacteria, Bifidobacterium longum, which is nonpathogenic and anaerobic, selectively localized to and proliferated in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors after systemic application. We further ascertained the tumor specificity of genetically engineered, as well as wild-type, Bifidobacterium longum. This is the first demonstration that Bifidobacterium longum can be utilized as a specific gene delivery vector for gene therapy on solid breast tumors.

Methyl-substituted diindolylmethanes as inhibitors of estrogen-induced growth of T47D cells and mammary tumors in rats.

Diindolylmethane (DIM) is formed by acid catalyzed dimerization of the phytochemical indole-3-carbinol, and both compounds inhibit formation and/or growth of mammary tumors in rodents. In this study, we have investigated the aryl hydrocarbon receptor (AhR) agonist activity and inhibitory AhR-estrogen receptor crosstalk induced by the following methyl-substituted DIMs: 1,1'-dimethyl-, 2,2'-dimethyl-, 5,5'-dimethyl-, 6,6'-dimethyl-, and 7,7'-dimethylDIM and 1,1',2,2'-tetramethylDIM. The six compounds bound to the rat cytosolic AhR in a transformation assay but, at concentrations < or = 10 microM, exhibited minimal to non-detectable AhR agonist or antagonist activities associated with CYP1A1 induction. In contrast, the methyl-substituted DIMs inhibited estrogen-induced T47D human breast cancer cell growth and the four most active compounds (1,1'-, 2,2'-, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM) inhibited one or more estrogen-induced responses in the 21-day-old female B6C3F1 mice at a dose of 100 mg/kg/day (X3). Induction of hepatic CYP1A1-dependent activity was not observed at this high dose. The antitumorigenic activity of these compounds was examined in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor model in which the DIM analogs were orally administered (by gavage in corn oil) at a dose of 1 mg/kg/day (X10). 1,1'-DimethylDIM, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM significantly inhibited mammary tumor growth, and this was not accompanied by changes in organ/body weights or histopathology. These studies demonstrate that methyl-substituted DIMs are selective AhR modulators (SAhRMs) with potential for clinical treatment of breast cancer.

A novel aromatase inhibitor, vorozole, shows antitumor activity and a decrease of tissue insulin-like growth factor-I level in 7, 12-dimethylbenz[a]anthracene-induced rat mammary tumors.

Effects of vorozole, a potent and specific non-steroidal aromatase inhibitor, were evaluated on female Sprague-Dawley (SD) rats with 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors. Vorozole at a dose of 0.25, 1.0 and 4.0 mg/kg was orally administered once a day for 28 consecutive days. A significant regression in tumor size was observed in each treated group at 1, 2, 3 and 4 weeks after the start of treatment compared with control group. Tissue insulin-like growth factor I (IGF-I) in the DMBA-induced tumors in each treated group significantly decreased in a dose-dependent fashion compared with control group. These results show the mechanism of vorozole in DMBA-induced rat mammary tumors.

Additive effects of medroxyprogesterone acetate and 5-fluorouracil derivative on 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors.

Chronic oral administration of 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil suppressed thymidylate synthetase (TS) gene expression followed by reduction of TS activity in rat mammary tumors induced with 7,12-dimethylbenz[a]anthracene. Addition of medroxyprogesterone acetate (MPA) to the anticancer drug caused an additional decrease in TS and thymidine kinase activities in the tumor growth and restoration of bone loss. These results suggest that the simultaneous administration of MPA and anticancer drugs causes increased inhibition of mammary tumor growth and also diminishes the bone loss.

Inhibition of 7,12-dimethylbenz[ a]anthracene-induced rat mammary tumor growth by aryl hydrocarbon receptor agonists

The antitumorigenic activities of 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), 8-methyl-1,3,6-trichlorodibenzofuran (8-MCDF) and 6-cyclohexyl-1,3,8-trichlorodibenzofuran (6-CHDF) were investigated in the 7,12-dimethylbenz[ a]anthracene (DMBA) rat mammary tumor model. At doses of 5, 10 or 25 mg/kg per week, both 6-MCDF and 8-MCDF significantly inhibited mammary tumor growth and at the 5 mg/kg per week dose >50% growth inhibition was observed. In contrast, 6-CHDF was inactive at the 5 mg/kg per week dose and the structure-antitumorigenicity relationships (6-MCDF/8-MCDF>6-CHDF) correlated with structure-antiestrogenicity (rat uterus) studies and the relative binding affinities of these compounds for the aryl hydrocarbon receptor (AhR). The antitumorigenic activity of 6-MCDF or 8-MCDF in the mammary was not accompanied by any significant changes in liver/body weight ratios, liver morphology or induction of hepatic CYP1A1-dependent activity which is one of the most sensitive indicators of exposure to AhR agonists. RT-PCR and Western blot analysis of mammary tumor mRNA and protein extracts, respectively, confirmed the presence of AhR suggesting that AhR-mediated signaling pathways are functional in rat mammary tumors. These results define a relatively non-toxic group of AhR agonists which exhibit potent antitumorigenic activity in the DMBA-induced rat mammary tumor model (<1 mg/kg per day), and therefore represent a new class of indirect-acting antiestrogens which have potential for clinical treatment of mammary cancer.

Estrogen regulates vascular endothelial growth/permeability factor expression in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

Estrogen regulates vascular endothelial growth/permeability factor expression in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

Estrogen regulates vascular endothelial growth/permeability factor expression in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

Vascular endothelial growth/permeability factor (VEG/PF) is expressed in some normal tissues and at high levels in a wide range of tumors. This growth factor is believed to be a key mediator of angiogenesis. Recent reports have shown that VEG/PF mRNA in the normal rat uterus is stimulated by estradiol (E2). In this study, we investigated the expression of VEG/PF in the mammary gland and 7,12-dimethylbenz(a)anthracene (DMBA)-induced, hormone-dependent mammary tumor of the rat model, and also whether VEG/PF is regulated by E2. VEG/PF mRNA from tumor extracts was amplified by RT-PCR with VEG/PF primers and generated two main products which corresponded in size to those expected for VEG/PF 164 and 120. In some cases, a third product corresponding in size to that expected for VEG/PF 188 was also generated. No such PCR products were generated from equal amount of RNA from normal mammary tissue, rat brain, or liver. Using immunocytochemistry, VEG/PF expression was detected in the epithelial cells of the tumors. We developed an ELISA assay to measure VEG/PF protein concentrations and found a 4-fold difference between normal mammary glands (1.3 +/- 0.11 ng/mg protein) and tumors (4.44 +/- 0.66) (P < 0.01). E2 treatment (5 microg/rat, s.c.) of rats 24 h after ovariectomy, greatly enhanced the expression of RT-PCR products in tumors within 2 h, which reached a maximum at 6-8 h but declined by 48 h. VEG/PF concentrations were also increased 8-12 h after E2 injection. When rats were given two injections of aromatase inhibitor 4-hydroxyandrostenedione (4-OHA 10 mg/rat s.c.) 24 h apart, to reduce estrogen concentrations, a low level of RT-PCR products was maintained for at least 96 h. After a single injection of 4-OHA, RT-PCR products remained low until 36 h when an increase occurred corresponding with a rise in plasma E2 levels. Injection of E2 2 h after 4-OHA treatment, caused a rise in RT-PCR products in 6-8 h. However, there was no significant change in VEG/PF concentrations. An increase in VEG/PF protein concentrations followed the increase in mRNA levels by 4-6 h. Thus, it appears that E2 causes a rapid induction of VEG/PF expression in mammary tumors that is similar to that observed in the normal uterus. These findings suggest that one mechanism by which estrogen acts as a mammary tumor promotor is by stimulating VEG/PF, leading to increased tumor angiogenesis and/or permeability of the microvessels to allow tumor cell migration.

Usefulness of 22-oxa-1,25-dihydroxyvitamin D-3 (OCT) as a single agent or combined therapy with aromatase inhibitor (CGS 16949A) on 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors

The antitumor effects of 22-oxa-1,25-dihydroxy-vitamin D-3 (OCT), a vitamin D-3 analogue, were evaluated on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumors. The combined effects of OCT (0.3 mu g/kg) with tamoxifen (0.5 mg/kg) medroxyprogesterone acetate (MPA) 2.5 mg/kg), or a new aromatase inhibitor, CGS 16949A (0.8 mg/kg) were also evaluated. OCT significantly suppressed the growth of tumors without hypercalcemia in a dose dependent manner at the fourth week from the start of treatment. Tumor size in the OCT+CGS 16949A group was significantly decreased compared with that in the OCT or CGS 16949A alone. However, there was no significant difference in tumor size between OCT alone and combined therapy with tamoxifen or MPA. We conclude that a single administration of OCT, which does not cause hypercalcemia, is effective for breast cancer and that a combination of OCT and aromatase inhibitor, CGS 16949A augments the antitumor effect on tumors compared to each single agent.