Inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis and of in vivo formation of mammary DMBA-DNA adducts by rosemary extract

The effect of dietary intake of an extract of the spice plant Rosmarinus officinalis L. on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis and on the in vivo formation of mammary DMBA-DNA adducts was evaluated. Supplementation of a semi-purified diet with 1.0% (by wt.) rosemary extract resulted in a significant (47%) decrease in mammary tumor incidence compared to controls. In subsequent studies, dietary supplementation with 0.5% and 1.0% rosemary extract inhibited total in vivo binding of DMBA to mammary epithelial cell DNA by an average of 42%. This decrease in total binding was not due to a uniform decrease in the formation of all mammary DMBA-DNA adducts. The formation of two major adducts derived from the anti-diastereomer of DMBA and bound to deoxyguanosine (anti-dGuo) was significantly decreased at both dietary rosemary concentrations. The formation of the syn-dGuo adduct also was inhibited, whereas formation of the syn-dAdo adduct was unaffected by consumption of the rosemary extract. These studies suggest that use of rosemary extract and its individual antioxidative constituents as chemopreventative agents for experimental mammary tumorigenesis warrant further investigation.     

Prevention of rat mammary carcinoma utilizing leuprolide as an equivalent to oophorectomy

A clinical trial is currently under way to examine the effectiveness of leuprolide as a breast cancer chemopreventive agent and contraceptive. This trial, as well as similar proposed studies, is based on the assumption that leuprolide is as effective as surgical castration in preventing the onset of mammary tumors; however, this has not been well documented in the DMBA animal model. We directly compared leuprolide and oophorectomy in this model and examined a combined therapy of leuprolide/bromocriptine. Twenty-seven day old female Sprague-Dawley rats were randomly allocated into one of eight groups. All rats received a 20-mg dose of DMBA at the age of 55 days. Group 1 (n=10), no treatment; Group 2 (n=9), leuprolide (100g/kg/day) for eight weeks beginning four weeks prior to DMBA; Group 3 (n=10), oophorectomy four weeks prior to DMBA with replacement estrogen beginning four weeks following DMBA. Estrogen replacement was achieved with a 0.05-mg estradiol tablet releasing 0.833g/day over a 60-day period. Group 4 (n=10), leuprolide (100g/kg/day) initiated two weeks prior to DMBA and continuing for two weeks following DMBA; Group 5 (n=9), oophorectomy two weeks prior to DMBA with 0.05mg of estradiol in depot form, releasing 0.833g/day, beginning four weeks following DMBA and continuing until week 16 of the study; Group 6 (n=10), leuprolide (100g/kg/day) beginning two weeks prior to DMBA and continuing for the duration of the experiment; Group 7 (n=10), leuprolide (100g/kg/day) for eight weeks beginning two weeks prior to DMBA; Group 8 (n=9), leuprolide (100g/kg/day) and bromocriptine (83g/day) for eight weeks beginning two weeks prior to DMBA. At nineteen weeks (15 weeks post DMBA), animals were sacrificed and autopsies performed. One hundred percent of untreated animals developed tumors. No animals undergoing oophorectomy four weeks prior to DMBA or receiving leuprolide four weeks prior to and simultaneously with DMBA developed tumors. In animals pretreated two weeks prior to DMBA with leuprolide or oophorectomy, each group had one animal with tumor development. No tumors developed in the animals receiving ongoing injections of leuprolide. However, one tumor developed in those receiving leuprolide for the first eight weeks beginning two weeks prior to DMBA administration. One animal receiving both leuprolide and bromocriptine developed one tumor. We conclude that chemical oophorectomy (with leuprolide) is as effective as surgical oophorectomy in inhibiting DMBA induced carcinogenesis.     

Black tea and mammary gland carcinogenesis by 7,12- dimethylbenz[a]anthracene in rats fed control or high fat diets

Epidemiological studies suggest that tea may reduce cancer risk, and in laboratory rodents, chemopreventive effects of tea or purified extracts of tea have been demonstrated in lung, gastrointestinal tract and skin. There is some evidence of chemoprevention by tea in the mammary gland, but the data are not conclusive. In order to evaluate more fully the possible influence of black tea on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland tumors in the female S-D (Sprague-Dawley) rat, three large studies were performed: experiment 1, tumorigenesis in rats fed AIN-76A diet and given 25 mg/kg DMBA and 1.25 or 2.5% whole tea extract or water to drink; experiment 2, tumorigenesis in rats given 15 mg/kg DMBA and the same diet and fluids as in experiment 1; experiment 3, tumorigenesis in rats fed control or HF (high fat, corn oil) diet and given 15 mg/kg DMBA and 2% tea or water to drink. Tea was given throughout the experiment; DMBA was given by gastric gavage at 8 weeks of age. There was no consistent effect of tea on tumorigenesis in rats fed AIN-76A diet; there was, however, evidence in experiment 3 of a reduction of tumorigenesis by tea in rats fed the HF diet. In experiment 3, rats fed the HF diet and given water showed the expected increase in tumor burden (number and weight) compared with rats fed control diet. However, rats fed the HF diet and given 2% tea showed no increase in tumor burden; their tumor burden was significantly lower than in rats fed the HF diet and given water (P < 0.01) and was not different from rats fed control diet and given water or tea. In addition, in experiment 3, the number of malignant tumors per tumor- bearing rat was increased by the HF diet in water-drinking rats (P < 0.01) but not in tea-drinking rats. Therefore, it appears that tea partially blocked the promotion of DMBA-induced mammary tumorigenesis by the HF diet.      

A case-control study of reproductive variables,alcohol, and smoking in premenopausal bilateral breast cancer

Summary The transforming growth factor-s are potent growth inhibitors of normal and transformed breast epithelial cells in culture. In vivo, these peptides modulate the development of the mouse mammary gland. Tissuespecific overexpression of mature TGF-1 in transgenic mice results in mammary gland atrophy and prevention of carcinogen-induced breast tumorigenesis. However, the inhibitory effect of endogenous or exogenous TGF-s on established tumor cells is less clear. Several published circumstantial and more direct data argue that, in some cases, the tumor cell TGF-s may contribute to the maintenance and/or progression of tumor cells in an intact host by modulating their interaction with host factors. This differential role of the TGF-s on mammary cells as determined by their normal or transformed phenotype as well as the biological and clinical implications of these data are discussed.Presented at the symposium "New Approaches in the Therapy of Breast Cancer", Georgetown University Medical Center, Washington DC, October 1994, generously supported by an education grant from Bristol-Myers Squibb.     

Adrenal steroids stimulate growth and progesterone receptor levels in rat uterus and DMBA-induced mammary tumors

We have studied the effect of treatment with the adrenal steroids androst-5-ene-3,17-diol (⁵-diol) and dehydroepiandrosterone (DHEA) on the growth and progesterone receptor levels of dimethylbenz-(a)anthracene (DMBA)-induced mammary tumors in the rat.While the total number of tumors in ovariectomized animals was 0.60 ± 0.19 tumor per rat after 24 days, it increased to 2.54 ± 0.50 (p<0.01) and 1.42 ± 0.26 (p<0.01) in the ⁵-diol and DHEA (2 mg, twice daily) treated animals, respectively. While very few new tumors developed during a 24-day period in ovariectomized animals (0.07 ± 0.07/rat), an average of 0.47 ± 0.19 (p<0.05) new tumor per animal appeared in intact rats. In ovariectomized animals treated with ⁵-diol or DHEA, the numbers of new tumors were 0.77 ± 0.26 (p<0.05) and 0.42 ± 0.15 (p<0.05), respectively. An even more striking effect was observed on average total tumor area, which decreased from 4.70 ± 0.95 cm² in intact animals to 0.75 ± 0.27 cm² (p<0.01) following ovariectomy. Values of 9.79 ± 2.25 (p<0.01) and 3.93 ± 0.86 cm² (p<0.01) were found in the ⁵-diol- and DHEA-treated ovariectomized animals, respectively.Treatment of ovariectomized animals with ⁵-diol and DHEA caused a marked increase (p<0.01) in progesterone receptor levels in both the uteri and DMBA-induced mammary tumors. Uterine weight was also stimulated (p<0.01) by treatment with the two adrenal steroids.The present data show that two adrenal C₁₉-⁵ steroids, ⁵-diol and DHEA, possess stimulatory effects analogous to those of estrogens on DMBA-induced mammary tumor growth and progesterone receptor levels in the rat, thus supporting the suggestion of an important role of these adrenal steroids in breast cancer and other estrogen-sensitive diseases in the human.     

A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CG conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CG-chain in vitro. To test the hypothesis that the Hecate-CG conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CG conjugate and Hecate alone. Cytotoxic effects of the Hecate-CG conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CG conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CG conjugate to LHR. At a concentration of 33 µM the conjugate inhibited (50%; IC₅₀) the binding of CG to LHR.We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CG for in vivo trials.     

Involvement of TGF-βs/TβRs System in Tumor Progression of Murine Mammary Adenocarcinomas

We studied the expression of TGF-/TR system and its biological role in tumor development, in M3 and MM3 murine mammary adenocarcinomas with different metastasizing capability and in LM3 and LMM3 derived cell lines. All the studied cells secreted TGF-s and expressed TRs. While the proliferation of the poorly metastatic M3 cells was significantly inhibited by 4 ng/ml TGF-s, the highly metastatic MM3 cells were only slightly inhibited in response to the highest dose used. LM3 and LMM3 cells, highly invasive and metastatic, were totally refractory to TGF- antiproliferative effect. The role of TGF- in modulating key proteolytic cascades in tumor progression was also studied. TGF-s enhanced metalloproteinases production in all the studied cells while induced a stimulatory net effect on plasmin system activity only in the more metastatic cells. Our results in this murine mammary tumor lineage support the concept that dissociation of TGF- regulated growth control versus proteolytic enzyme pathways promotes tumor dissemination.     

Sex hormone-induced mammary carcinogenesis in female Noble rats: detection of differentially expressed genes

Breast cancer is the most common cancer and the second most frequent cause of cancer death in women. Epidemiological data has recognized that an increased cumulative exposure to estrogen is the common tie linking most of the established risk factors for breast cancer. Sex hormone-induced mammary gland carcinogenesis of the Noble rat (using testosterone and 17-estradiol) resembles that of the human counterpart in its growth pattern as well as the histopathology of the tumors induced. This model may provide a paradigm for examination of genetic alterations and changes in gene expression between different histological groups and to make inferences about the role of known and putative oncogenes and tumor suppressor genes. We studied the gene expression profile during sex hormone-induced mammary carcinogenesis using a cDNA array technique; the results were further confirmed by RT-PCR, western blotting and immunohistochemical analyses. From the 10 differentially expressed genes identified, we have studied four highly overexpressed genes, two cell cycle/growth control regulators, the cyclins D1 and D2, a growth factor, IGF-2 and a cytokine TNF-. Cyclins D1 and D2 were highly expressed in the nuclei of carcinoma cells but at low levels in the nuclei of the hyperplastic and normal mammary tissue. IGF-2 was found to expressed in the cytoplasm of the carcinoma cells but not in the stromal cells. Western blot showed expression of big IGF-2 consistent with the tumor derived truncated forms of pro-IGF-2. The matured circulating IGF-2 at 7.5 kDa identified in the serum was not expressed in any of the breast tissue samples. TNF- expression was found not only in the macrophages but also in the mammary carcinoma cells. The result of the present study provides some information on the molecular basis of this sex hormone-induced mammary carcinogenesis and the role of these proteins in tumor progression.     

Inhibitory effects of medroxyprogesterone acetate (MPA) and the pure antiestrogen EM-219 on estrone (E1)-stimulated growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat

Summary Estrogens are well known to play a predominant role in promoting the growth of DMBA-induced mammary tumors in the rat. Estrone (E₁), a steroid having weak estrogenic activity, is one of most important estrogens in post-menopausal women, where it is converted into the potent estrogen estradiol (E₂) by 17-hydroxysteroid dehydrogenase (17-HSD) in many peripheral tissues, including the mammary gland. In this report, we have studied the effect of a new antiestrogen (EM-219) (N-butyl, N-methyl-11-(3, 17-dihydroxy-17-ethinylestra-135(10), 14-tetraen-7-yl) undecanamide) on E₁-stimulated growth of DMBA-induced mammary tumors and compared its effect with that of medroxyprogesterone acetate (MPA) alone or in combination. After 18 days, ovariectomy (OVX) reduced total tumor area to 29.6 ± 7.1% of the original size, while E₁ (1.0 µg, twice daily) caused a 139 ± 21% increase in tumor size in OVX animals. MPA (1.5 mg, twice daily) partially reversed the stimulatory effect of E₁ to 66.0 ± 9.0%, while the antiestrogen EM-219 (40 µg, twice daily) decreased tumor size to 70.0 ± 10%. Combination of these two compounds led to a further inhibition of tumor size to 30.7 ± 7.4% of the value found in OVX animals treated with E₁. Tumor E₂ levels decreased from 1688 ± 155 pmoles/kg tissue in OVX animals receiving E₁ to 709 ± 92, 1347 ± 98, and 184 ± 11 pmoles/kg tissue in MPA-, EM-219-, and MPA + EM-219-treated OVX-E₁ animals, respectively. Treatment of OVX animals with E₁ increased by 69% the reductive activity of 17-hydroxysteroid dehydrogenase (17-HSD) while MPA abolished completely this effect of E₁. In the oxidative direction, treatment with E₁, E₁ + MPA, or E₁ + EM-219 had minimal or no significant effect on the activity of 17-HSD (vs OVX), while the combined treatment with MPA + EM-219 induced a 2-fold increase in 17-HSD activity, thus leading to an increased conversion of E₂ into E₁. The present data show that combination of the pure antiestrogen EM-219 with MPA exerts a greater reduction in DMBA-induced mammary tumor growth and intratumoral E₂ levels stimulated by E₁ than either compound used alone. This interactive effect of the antiestrogen and MPA could at least partially be related to the increased inactivation of E₂ into E₁. The present data suggest that such a combination could be a useful approach for the treatment of breast cancer, especially in post-menopausal women.     

Mammary tumorigenesis in growth hormone deficient spontaneous dwarf rats; effects of hormonal treatments

This study was carried out to investigate mammary tumorigenesis in growth hormone (GH) deficient spontaneous dwarf rats (SDR). At 50–60 days of age, the rats were divided into five groups. Group 1 received bovine (b) GH (prolonged release formulation) administered at a dose of 40–50 mg/kg body wt. in 50 l weekly injections; group 2 received recombinant human insulin-like growth factor-I (IGF-I) at a dose of 1 mg/kg body wt./day administered via osmotic pumps; animals in group 3 were fitted with subcutaneous silastic capsule containing 30 g 17-estradiol (E2) plus 30 mg progesterone (P4), replaced every 2 months; group 4 received both bGH and E2 plus P4 treatments at the same doses as above, and control animals (group 5) received sham treatments (vegetable oil injection, silastic capsules containing cellulose). After 1week of treatment, all animals were injected intraperitoneally with the carcinogen N-methyl-N-nitrosourea (MNU) at a dose of 50 mg/kg body wt. Other groups of animals, receiving identical hormonal treatment to those exposed to MNU, were treated for 10 days only and then sacrificed for assessment of circulating concentrations of hormones and mammary gland characteristics at the time of carcinogen exposure. The hormonal treatments of the animals exposed to the MNU were continued for an additional 20 weeks and mammary tumor development monitored by weekly palpation and tumors collected as necessary. The rats were weighed weekly. At the end of the treatment period, all animals were sacrificed and remaining tumors were collected. Rats in all groups continued to gain weight throughout the experimental period, but the largest weight gain was see in animals receiving GH either alone or with E2 and P4. Animals treated with IGF-I also gained weight compared to controls, but this weight gain was less than that seen in GH-treated rats. GH treatment alone increased mammary tumor incidence from 4.8% in controls to 100%. Average tumor load and latency in the GH-treated rats were 7.0 ± 0.8 tumors/tumor-bearing rat (mean±SEM) and 57.3 ± 2.7 days (mean±SEM), respectively. As in intact Sprague–Dawley rats, approximately 90% of the tumors that developed in the GH-treated rats were ovarian dependent for growth. IGF-I treatment also increased mammary tumor development to 62.5%. Average tumor load and latency in the IGF-I-treated rats were 1.6 ± 0.4 tumors/tumor-bearing rat (mean±SEM) and 96.2 ± 14.5 days (mean±SEM), respectively. However E2+P4 treatments did not significantly alter tumorigenesis and, surprisingly, simultaneous treatment with E2+P4 and GH obliterated the GH-stimulated increase in tumor development. Prolactin (PRL) did not appear to influence mammary tumorigenesis in the SDRs, as untreated SDRs had significantly elevated serum concentration of PRL as compared with normal Sprague–Dawley (SD) rats, whereas GH-treated SDRs had PRL levels similar to that of normal SD rats. No obvious structural characteristics were associated with high or low susceptibility to mammary tumorigenesis, as assessed by mammary gland whole mounts from the different animal groups sacrificed at the time of carcinogen administration.Enhanced expression of the extracellular signal-regulated kinase 1/2 (ERK1/2), and activation (phosphorylation) of ERK1/2 were associated with an increase in mammary tumorigenesis. Similarly, the expression of the estrogen receptor- (ER) was significantly elevated in animal groups with the highest susceptibility to tumorigenesis, whereas the levels of cyclin D1 expression were not related to mammary tumorigenesis.     

Sex hormone-induced mammary carcinogenesis in female Noble rats: Expression of TGF-β1 and its receptors, TGF-α, and EGF-R in mammary carcinogenesis

We have established a Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis, in which the role of androgen in promoting mammary carcinogenesis was highlighted. We have also established that stromal–epithelial interactions may be responsible for the promotional effects of testosterone in mammary carcinogenesis. Based on these understandings, in the present study we examined the expression of transforming growth factor beta-1 (TGF-₁) and its receptors (TGF- RI, TGF- RII), transforming growth factor alpha (TGF-), and epidermal growth factor receptor (EGF-R) in 'pre-malignant' mammary glands treated with different protocols of sex hormones, as well as in mammary cancers. We observed that TGF-₁ was strongly expressed in most mammary tumors, whereas TGF- RI and TGF- RII were negative in most mammary tumor cells. The results from comparative study of 'pre-malignant' glands further showed that when the animals were treated with testosterone, either alone or in combination with 17-estradiol, the mammary gland epithelial cells expressed high levels of TGF-₁. This over-expression of TGF-₁ can be blocked by flutamide, indicating that testosterone may be responsible for the expression of TGF-₁ in mammary glands. TGF- RI and TGF- RII were also expressed strongly in testosterone-treated mammary epithelial cells and only weakly detectable in 17-estradiol treated and control mammary epithelial cells. Furthermore, TGF- RI and TGF- RII were also expressed in stromal cells, both in mammary tumors and in hormone-treated mammary glands. These observations indicate that the mechanism of testosterone in mammary carcinogenesis may be through its regulation of expression of TGF-₁ and its receptors. On the other hand, TGF- was also expressed in all 39 mammary cancers, while only 81% of the cancers were EGF-R positive. TGF- was also strongly expressed in stromal cells in all three experimental groups, but only moderately expressed in epithelial cells when treated with a combination of testosterone and 17-estradiol. By contrast, EGF-R was strongly expressed in epithelial cells in the three experimental groups but negative in stromal cells. Flutamide or tamoxifen was unable to block the expression of TGF- induced by the combined sex hormone treatment. However, they were effective in blocking the expression of TGF- when the animals were treated with testosterone or 17-estradiol alone, respectively. These results suggest that both testosterone and 17-estradiol may be required for the over-expression of TGF- in the mammary carcinogenesis induced by sex hormones. To our knowledge, this is the first experimental study to explore the regulation of TGF-₁, TGF-, and their receptors by testosterone and 17-estradiol in mammary carcinogenesis.     

Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells

Summary The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulationsin vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E₂) metabolites onin vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E₂ via the C2- and C16-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E₂ metabolites. A single 24 hr exposure of 0.78 µM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16 hydroxylation of E₂. The DMBA treated C57/MG cells also exhibited increased anchorage-independencein vitro prior to tumorigenesisin vivo. A simultaneous treatment of cells with DMBA and with the highest non-cytotoxic doses of the tumor suppressing agents 5 µM N-(4-hydroxyphenyl) retinamide (HPR), 50 µM indole-3-carbinol (I3C), or 1 µM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16 hydroxylation of E₂, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E₂ metabolites at the highest non-cytotoxic doses of 0.76 µM estrone (E₁), 0.69 µM 2-hydroxyestrone (2-OHE₁), and 0.66 µM 2-methoxyestrone (2-MeOHE₁) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E₂ metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16 hydroxylation in favor of C16-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E₂ metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HPR N-(4-hydroxyphenyl) retinamide - I3C indole-3-carbinol - TAM tamoxifen - E₂ 17-estradiol - E₁ estrone - 2-OHE₁ 2-hydroxyestrone - 2-MeOHE₁ 2-methoxyestrone - 16-OHE₁ 16-hydroxyestrone - E₃ estriol - DME/F12 Dulbecco's modified Eagle's medium - F12 Ham's medium - HU hydroxyurea - PBS phosphate buffered saline - NaOH sodium hydroxide - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - [C2-³H] E₂ estradiol labeled at C2 position - [C16-³H] E₂ estradiol labeled at C16 position - ANOVA analysis of variance     

Inhibitory action of ASPARAGUS racemosus on DMBA-induced mammary carcinogenesis in rats

The present paper reports the inhibitory effect of Asparagus racemosuson DMBA-induced mammary tumorigenesis in rats of Holtzman strain. When virgin female rats, normal or primed with 17-β-estradiol treatment, are put on diets containing 0.25%, 0.5%, 1% or 2% Asparagus root extract powder prior to their exposure to DMBA, mammary tumor incidence shows a sharp decline.     

Adrenal regulation of mammary tumorigenesis in female Sprague-Dawley rats: incidence, latency, and yield of mammary tumors

Huggins and Morii (J. Exp. Med., 114: 741, 1961) reported that massive adrenal necrosis occurs in 79 and 100% of female Sprague-Dawley rats receiving 20 and 30 mg, respectively, of the mammary carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Here, adrenal necrosis and regeneration were studied in 158 rats for up to 21 days post-DMBA by radioautography of the adrenals of animals given 50 microCi [3H]thymidine 30 min before sacrifice. Adrenal cell proliferation was markedly inhibited 21 days post-DMBA. Regenerated adrenals were more susceptible to this adrenocorticolytic effect. To investigate if alterations in adrenal function modify tumorigenesis, animals underwent adrenalectomies (ADX), hypophysectomies, ovariectomies, and pituitary transplants alone or in combination 6 days after receiving DMBA (20 mg/100 g intragastrically) at 50 days of age. To prevent adrenal necrosis, 24 animals were pretreated with metyrapone. Methylprednisolone acetate, 1 mg i.m., was given to 40 animals every 5 days beginning 6 days post-DMBA. There were 50 non-DMBA-treated intact and surgical controls. DMBA was necessary but not sufficient to induce mammary tumors. No tumors developed in controls or in 46 animals hypophysectomized 6 days after DMBA. Metyrapone reduced tumor incidence and yield. ADX after DMBA treatment increased the tumorigenic response and eliminated resistance to tumorigenesis in older rats. Only three tumors developed in DMBA-treated rats receiving methylprednisolone acetate. Mammary tumorigenesis was increased by pituitary transplant 6 days after DMBA to intact and ADX animals. Ovariectomy 6 days after DMBA was as effective as methylprednisolone acetate in preventing tumorigenesis; ADX did not overcome either inhibition. We conclude that adrenal hormones inhibit proliferation of initiated mammary cells.     

Growth factor involvement in the multihormonal regulation of MCF-7 breast cancer cell growth in soft agar

Summary The hormone dependency of the MCF-7 breast cancer cell line, while extensively tested in liquid culture, has not been previously evaluated under conditions of anchorage-independent growth in serum-free media. Using the soft agar clonogenic assay, we demonstrate that physiologically relevant concentrations of estradiol (E₂), progesterone (Pg), and prolactin (PRL) similarly stimulated MCF-7 cell colony formation in the absence of serum. Addition of an anti-insulin-like growth factor-I (IGF-I) antibody inhibited E₂- and Pg-stimulated growth, while PRL action was not affected. Similar results were obtained with an anti-IGF-I receptor antibody, except that its inhibitory effect on Pg-induced colony formation was modest and not statistically significant. Administration of either an anti-transforming growth factor- (TGF-) antibody or an anti-epidermal growth factor (EGF) receptor antibody similarly inhibited E₂-stimulated MCF-7 cell growth in soft agar, while neither antibody influenced Pg or PRL effects. Addition of TGF-₁, -₂, -₃ similarly suppressed MCF-7 cell colony formation in a dose dependent manner to a degree comparable to that observed with 4-OH-tamoxifen (4-OH-T). Furthermore, the growth inhibitory effect of 4-OH-T was completely reversed by an anti-TGF- antibody. We conclude that IGFs and TGF- are important mediators of E₂-stimulated MCF-7 cell growth in soft agar. IGFs may also be playing a role in Pg action, while neither growth factor is involved in PRL-stimulated colony formation. Finally, TGF- appears to be an important mediator of antiestrogen-induced inhibition of tumor growth.Abbreviations IGF insulin-like growth factor - TGF transforming growth factor - E₂ estradiol-17 - Pg progesterone - PRL prolactin - 4-OH-T 4-hydroxy-tamoxifen - IMEM improved minimal essential medium     

Tumorigenesis of mammary gland by 7,12-dimethylbenz(a)anthracene during pregnancy: relationship with DNA synthesis

The relationship between mammary cell proliferation during pregnancy and susceptibility to 7,12-dimethylbenz(a)anthracene (DMBA) was examined. DMBA was administered intravenously to Sprague-Dawley rats on the 5th, 10th or 15th day of pregnancy. [3H]thymidine labelling index (LI) of the mammary cells at the time of treatment with the carcinogen was determined and found to be higher in the pregnant rats than in age-matched virgin controls. In spite of the high proliferative index of the mammary cells, significant inhibition of tumorigenesis occurred in the pregnancy rats allowed to complete pregnancy and parturition following treatment with DMBA. However, when pregnancy was terminated by cesarian section shortly after treatment with DMBA, there was a significantly higher tumor incidence as compared to the "full-term" rats. It was observed that the earlier the pregnancy was terminated, the greater was the incidence of mammary tumors. This would indicate that the inhibitory effect of pregnancy is related to changes occurring during the later half of gestation. The differentiation of mammary cells for milk synthesis as pregnancy progresses is postulated to be a major reason for the observed refractoriness of the mammary cells to DMBA at that time.     

Pubertal high fat diet: effects on mammary cancer development

Introduction

Epidemiological studies linking dietary fat intake and obesity to breast cancer risk have produced inconsistent results. This may be due to the difficulty of dissociating fat intake from obesity, and/or the lack of defined periods of exposure in these studies. The pubertal mammary gland is highly sensitive to cancer-causing agents. We assessed how high fat diet (HFD) affects inflammation, proliferative, and developmental events in the pubertal gland, since dysregulation of these can promote mammary tumorigenesis. To test the effect of HFD initiated during puberty on tumorigenesis, we utilized BALB/c mice, for which HFD neither induces obesity nor metabolic syndrome, allowing dissociation of HFD effects from other conditions associated with HFD.

Methods

Pubertal BALB/c mice were fed a low fat diet (12% kcal fat) or a HFD (60% kcal fat), and subjected to carcinogen 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumorigenesis.

Results

HFD elevated mammary gland expression of inflammatory and growth factor genes at 3 and 4 weeks of diet. Receptor activator of nuclear factor kappa-B ligand (RANKL), robustly induced at 4 weeks, has direct mitogenic activity in mammary epithelial cells and, as a potent inducer of NF-κB activity, may induce inflammatory genes. Three weeks of HFD induced a transient influx of eosinophils into the mammary gland, consistent with elevated inflammatory factors. At 10 weeks, prior to the appearance of palpable tumors, there were increased numbers of abnormal mammary epithelial lesions, enhanced cellular proliferation, increased growth factors, chemokines associated with immune-suppressive regulatory T cells, increased vascularization, and elevated M2 macrophages. HFD dramatically reduced tumor latency. Early developing tumors were more proliferative and were associated with increased levels of tumor-related growth factors, including increased plasma levels of HGF in tumor-bearing animals. Early HFD tumors also had increased vascularization, and more intra-tumor and stromal M2 macrophages.

Conclusions

Taken together in this non-obesogenic context, HFD promotion of inflammatory processes, as well as local and systemically increased growth factor expression, are likely responsible for the enhanced tumorigenesis. It is noteworthy that although DMBA mutagenesis is virtually random in its targeting of genes in tumorigenesis, the short latency tumors arising in animals on HFD showed a unique gene expression profile, highlighting the potent overarching influence of HFD.     

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Transforming growth factor-β in breast cancer: A working hypothesis

Transforming Growth Factor- (TGF) is the most potent knowninhibitor of the progression of normal mammary epithelial cells through thecell cycle. During the early stages of breast cancer development, thetransformed epithelial cells appear to still be sensitive toTGF-mediated growth arrest, and TGF can act as an anti-tumorpromoter. In contrast, advanced breast cancers are mostly refractory toTGF-mediated growth inhibition and produce large amounts of TGF,which may enhance tumor cell invasion and metastasis by its effects onextracellular matrix. We postulate that this seemingly paradoxical switch inthe responsiveness of tumor cells to TGF during progression is theconsequence of the activation of the latent TGF that is produced anddeposited into the tumor microenvironment, thereby driving the clonalexpansion of TGF-resistant tumor cells. While tumor cells themselvesmay activate TGF, recent observations suggest that environmental tumorpromoters or carcinogens, such as ionizing radiation, can cause stromalfibroblasts to activate TGF by epigenetic mechanisms. As thebiological effects of the anti-estrogen tamoxifen may well be mediated byTGF, this model has a number of important implications for the clinicaluses of tamoxifen in the prevention and treatment of breast cancer. Inaddition, it suggests a number of novel approaches to the treatment ofadvanced breast cancer.     

Influence of underfeeding during the "critical period" or thereafter on carcinogen-induced mammary tumors in rats

Normal cycling virgin female Sprague-Dawley rats were divided into different treatment groups and subjected to half ad libitum feed intake for 2 or 4 weeks at consecutive periods of time before and/or after administration of 7,12-dimethylbenz(a)anthracene (DMBA) and then were returned to full-feed. All rats at the end of their respective under-feeding periods showed reductions in serum prolactin and cessation of estrous cycles. However, only rats underfed 1 week prior to and 1 week after DMBA administration showed significant reductions in mammary tumorigenesis or the entire 21 weeks of the experiment when compared to full-feed controls. Rat groups underfed during subsequent weeks after DMBA administration showed no alterations in mammary tumor development as compared to full-fed controls. These results demonstrate that inhibition and perhaps permanent suppression of mammary tumorigenesis occurred only in rats underfed 1 week before and 1 week after DMBA administration. The inhibitory effects produced by this under-feeding regimen on mammary tumorigenesis may be mediated by suppression of prolactin and estrogen secretion.