Effect of human urinary extract on initiation and growth of spontaneous murine mammary tumours

A material with gonadotrophin-inhibiting properties was extracted from pooled human urine of male subjects. This material was able to prevent initiation of mammary tumour growth in C3H(Jax) mice. When injected into tumour-bearing animals, 5 out of 31 tumours regressed while 21 out of 31 tumours stopped growing.     

Anti-carcinogenic activity of d-limonene during the initiation and promotion/progression stages of DMBA-induced rat mammary carcinogenesis

d-Limonene was found to be effective in reducing the averagenumber of rat mammary carcinomas that developed in 7,12-dimeihylbenz[a]anthracene-treatedrats when the terpene was fed during the initiation or duringthe promotion/progression stage of carcinogenesis. The timeto the appearance of the first tumor was extended only whend-limonene was fed during the initiation stage. These effectscould not be attributed to changes in mammary-relevant endocrinefunctions.     

Choline enhances acetylaminofluorene promotion of liver carcinogenesis in female but not in male rats.

The growth of focal lesions during chemical carcinogenesis has been analyzed after promotion in the liver of female and male rats receiving choline for 4 or 5 weeks. The number and size of foci of altered hepatocytes were first evaluated after methylnitrosourea was used as initiator and 2-acetylaminofluorene (2-AAF) associated with carbon tetrachloride as promoter. The development of enzyme-altered foci was lower in female rats than in males. Choline given intragastrically stimulated the growth of focal lesions in female rats by increasing both the enzyme-positive area of the single focus and the total area of positive foci per cm2 of liver section up to levels close to those of males. No effect of choline was observed in males. In a second set of experiments, focal proliferative lesions induced with the Solt-Farber model were larger in females fed a choline-enriched diet during the promotion phase, as a result of the area of the focus and the total area of foci per cm2 of liver being higher than that in females not receiving choline, with no significant change in the number of foci. When cell proliferation was selected during promotion by 2-AAF and carbon tetrachloride instead of partial hepatectomy, the effect of the choline-enriched diet was even more pronounced. This difference might be accounted for by the greater hepatotoxicity of xenobiotics after choline administration. The hypothesis that choline may enhanced hepatocyte susceptibility towards 2-AAF action by an increase in drug toxicity is discussed.     

Fasting/re-feeding before initiation enhances the growth of aberrant crypt foci induced by azoxymethane in rat colon and rectum

In contrast to the protective effect of chronic caloric restriction on tumor development, we have shown that fasting sustained tumor initiation in rat liver by a non-initiating dose of diethylnitrosamine. Here we investigated whether fasting had a similar favorable effect on initiation in the colorectal mucosa in 80 male F344 rats. Animals fasted for 4 days were given a single s.c. dose of azoxymethane (AOM) (20 mg/kg) on the first day of re-feeding, and rates of kinetic proliferative parameters, and development of the pre-neoplastic lesions such as aberrant crypt foci (ACF), were evaluated. Starvation before AOM treatment enhanced the growth of ACF, as shown by the significantly higher crypt multiplicity of fasted/re-fed rats as compared with fully fed rats (3.97 ± 0.50 vs. 2.64 ± 0.20, p ≤ 0.025). This difference was associated with perturbations in cell death and cell proliferation. Fasting induced apoptosis and depressed cell division, while re-feeding had opposite effects, resulting in a higher percentage of S-phase cells at the time of AOM injection and 2 days thereafter. Starvation-induced apoptosis may represent the mitogenic stimulus to an increase in the number of cells susceptible to AOM damage, and may favor its fixation, leading to enhanced growth of ACF. Our data therefore suggest that fasting/re-feeding enhances colon cancer. Int. J. Cancer 77:286–294, 1998.© 1998 Wiley-Liss, Inc.     

Overexpression of vascular endothelial growth factor-A165 enhances tumor angiogenesis but not metastasis during beta-cell carcinogenesis.

The pivotal role of vascular endothelial growth factor (VEGF-A) in the regulation of angiogenesis, in particular in the onset and maintenance of tumor angiogenesis, has been demonstrated repeatedly in experimental model systems and, more recently, in clinical trials. Experimental evidence has also suggested that up-regulated expression of VEGF-A may cooperate with other genetic or epigenetic changes to induce or accelerate tumor progression to invasive and metastatic cancers. Here we report the generation of transgenic mouse lines that express human VEGF-A165 under the control of the rat insulin promoter in the beta cells of pancreatic islets of Langerhans (Rip1VEGF-A). These mice do not exhibit detectable changes in islet development, vascularization, or physiology. Intercrosses of these mice with a transgenic mouse model of pancreatic beta cell carcinogenesis (Rip1Tag2) result in an earlier onset of tumor angiogenesis and with it accelerated tumor growth and mortality. The transition from benign tumors (adenoma) to malignant tumors (carcinoma) is modestly accelerated; however, tumor metastases are not observed. Our findings indicate that in beta-cell tumorigenesis, overexpression of VEGF-A165 accelerates the onset of tumor angiogenesis and with it tumor progression but is not sufficient to induce tumor metastasis.     

Suppression by beta-carotene-rich algae Dunaliella bardawil of the progression, but not the development, of spontaneous mammary tumours in SHN virgin mice

We previously found that beta-carotene-rich algae Dunaliella bardawil markedly inhibited spontaneous mammary tumourigenesis of mice. This study was carried out to clarify whether D. bardawil inhibits the development or the progression or both of mammary tumours. A high mammary tumour strain of SHN virgin mice were given vitamin A deficient AIN-76TM diet supplemented with D. bardawil during the limited period of 3 months between 1-4 months of age (Experiment I: the stage of tumour development), 4-7 months (Experiment II: the stage of initiation and progression) or 7-10 months (Experiment III: the stage of progression). The concentration of beta-carotene in the diet was 5.1 x 10(-5)%. The respective controls received AIN-76TM diet containing retinyl palmitate (2.2 x 10(-4)%) during the same periods as in the experimental groups. Both the experimental and the control mice were fed a commercial diet during all other periods. The diets and tap water were provided ad libitum. In Experiment I, mammary tumour incidence was higher in the experimental group than in the control at all months examined except at 5 months of age, while the cause is not clear at present. Meanwhile, mammary tumourigenesis was significantly suppressed in the experimental mice compared to the controls in Experiments II and III. Whereas tumorous mice were higher than non-tumorous mice in blood levels of lactic acid and glucose in the control, mice given D. bardawil maintained the levels of non-tumorous mice even after the development of tumours. D. bardawil feeding also induced a higher glucose tolerance. All results strongly suggest that D. bardawil can inhibit the progression of spontaneous mammary tumours of mice by increasing the homeostatic potential of the host animals as well as by the well-known antioxidant function of beta-carotene in D. bardawil.     

TIMP-3 deficiency in the host, but not in the tumor, enhances tumor growth and angiogenesis

Tumor cells, stromal cell compartment and the extracellular matrix (ECM) together generate a multifaceted tumor microenvironment. Matrix metalloproteinases and their tissue inhibitors (TIMPs) provide a means for tumor-stromal interaction during tumorigenesis. Among TIMPs, TIMP-3 is uniquely localized to the ECM and is frequently silenced in human cancers. Here, we asked whether the absence of TIMP-3 in the tumor cell or the host affects the process of tumorigenesis. Timp-3(-/-) ES-cell clones were generated and used to develop teratomas in nude mice. Timp-3(-/-) teratomas showed similar tumor take, growth, and angiogenesis compared to timp-3(+/+) teratomas. To study the effect of TIMP-3 ablation in the host stroma, we measured the growth kinetics of subcutaneous B16F10 melanomas in timp-3(-/-) and wild-type littermates. Tumors grew significantly faster in timp-3(-/-) than in wild-type mice and their CD31 content was significantly higher indicating increased angiogenesis. Augmented angiogenesis in timp-3(-/-) mice was directly tested using Matrigel plug and Gelfoam assays. In response to FGF-2, timp-3(-/-) endothelial cells invaded more efficiently, leading to enhanced formation of functional blood vessels. Thus, TIMP-3 deficiency in the host, but not in the tumor per se, leads to enhanced tumor growth and angiogenesis. TIMP-3 located within the tumor microenvironment inhibits tumorigenesis.     

Role of oncogene activation during prenatal (transplacental) initiation and postnatal promotion of mouse skin tumours

The transplacental initiation-postnatal promotion model of mouse skin carcinogenesis is useful in studying the molecular and cellular mechanisms of perinatal carcinogenesis. Offspring transplacentally exposed to an initiating dose of a carcinogen typically do not produce any skin tumours in the absence of postnatal treatment; many skin tumours appear only when they are treated with tumour-promoting agents postnatally. Tumour-promoting agents alone produce no skin tumours or only a few. Thus, two stages of carcinogenesis, initiation and promotion, can be conveniently separated. Our results indicate that fetal c-Ha-ras can be transplacentally activated through a specific point mutation by a carcinogen. However, since postnatal promotion was essential for the production of tumours, they also suggest that a cell harbouring such a mutation may remain dormant until it encounters a tumour-promoting stimulus. Since a higher fraction of carcinomas than papillomas contained the specific mutation in Ha-ras, it is postulated that those papillomas with the point mutation have a selective advantage to progress towards carcinomas.     

Interleukin-12 inhibits angiogenesis and growth of transplanted but not in situ mouse mammary tumor virus-induced mammary carcinomas

We examined the ability of recombinant murine interleukin-12 (rmIL-12) to inhibit the vasculature and growth of mammary carcinomas arising in situ in mouse mammary tumor virus (MMTV)-infected female C3H/HeN mice. Although it is a potent antiangiogenic and antitumor agent in many transplanted murine tumor models, rmIL-12 failed to inhibit the vascularity, reduce the perfusion, or alter the growth of these autochthonous carcinomas. Factors intrinsic to these tumor cells were unlikely to be responsible for therapy failure. This is because primary cells derived from these carcinomas responded to IFN-gamma, and rmIL-12 was effective against transplanted tumors arising from Mm5MT cells, a line established from a MMTV-induced mammary carcinoma in C3H mice. Factors intrinsic to the mice that host the autochthonous mammary carcinomas were also not responsible for failure, because they sponsored rmIL-12 antiangiogenic and antitumor effects against transplanted K1735 murine melanoma tumors. Instead, the autochthonous nature of the mammary carcinomas and their possession of a high percentage of mature, pericyte-covered vessels that are resistant to therapeutic regression may be responsible. This is supported by the observation that transplanted Mm5MT tumors had a lower proportion of pericyte-covered vessels and responded to rmIL-12 therapy. These results point to significant differences between the vasculature of transplanted and autochthonous murine tumors and indicate that their susceptibility to antivascular therapy may differ substantially.     

Recombinant lymphotoxin enhances the growth of normal, but not of chronic myeloid leukemic, human hematopoietic progenitor cells in vitro

The effects of recombinant lymphotoxin (rLT) and tumor necrosis factor (rTNF) on the growth of clonogenic normal and leukemic hematopoietic cells were investigated. Two opposite and dose-dependent effects of rLT on normal CFU-GM were found. Low concentrations (5pM) did stimulate the growth, while higher amounts of rLT showed an antiproliferative effect. In contrast, the effect of rTNF was only an inhibition of growth in a dose-dependent fashion. In chronic myeloid leukemia (CML), the CFU-GM was resistant to the growth stimulatory effect of rLT. Furthermore, CML-cells were found to be more susceptible than normal CFU-GM to the antiproliferative effect of both rLT and rTNF. These results show that LT and TNF exhibit qualitative differences in the effects on hematopoietic cells and that CML-cells display an increased susceptibility for the cytostatic effects on LT and TNF.     

Cyclic nucleotide levels in mouse mammary epithelial cells during growth arrest and growth initiation in culture.

Intracellular levels of cyclic AMP (cAMP) and cyclic GMP (cGMP) were measured in high and low tumorigenic mouse mammary epithelial cells during growth arrest in 1% fetal bovine serum and during the first 60 minutes after serum stimulation of cell proliferation in arrested cultures. Stationary MCG-T14 cells, which are highly tumorigenic and grow to high densities in 1% serum, exhibited lower levels of cAMP, higher levels of cGMP, and a lower ratio of cAMP to cGMP than quiescent MCG-V14 cells, which have low tumorigenicity and achieve low cell densities in 1% serum. Within 5-10 minutes after cell growth was initiated in arrested cultures by the addition of serum, both cell lines responded with a fourfold to fivefold increase in cGMP and a concomitant 50% decrease in cAMP. MCG-T14 cells exhibited the highest intracellular levels of cGMP and the lowest cAMP to cGMP ratio within 10 minutes after serum addition.     

Influence of the alkyllysophospholipid ET-18-OCH3 on methylnitrosourea-induced rat mammary carcinomas

This study describes the efficacy of the alkyllysophospholipid 1-octadecyl-2-methoxy-Sn-racglycero-3-phosphocholine (ET-18-OCH3) in inhibiting the growth of methylnitrosourea-induced mammary carcinomas in Sprague-Dawley rats. In experiment A 2 X 10 mg/kg Et-18-OCH3 were administered daily for 10 weeks prior to manifestation of mammary carcinomas which resulted in a significant inhibition of median tumor number and median tumor volume per rat. Treatment of established tumors (experiment B) with 6 and 60 mg/kg ET-18-OCH3 daily for 3 weeks effected a stagnation in tumor growth for the higher dosage only with 90% tumor inhibition in comparison to untreated controls; at the same time, however, clear toxic effects were seen, thus indicating a narrow therapeutic index of ET-18-OCH3 in single-drug therapy. Combination of ET-18-OCH3 with compounds possessing a different toxicity spectrum is suggested.     

Fate of 7,12-dimethylbenz(a)anthracene in the mouse mammary gland during initiation and promotion stages of carcinogenesis in vitro

Metabolic conversion and distribution of the products of 7,12-dimethylbenz(a)anthracene (DMBA) were analyzed in the mouse mammary cell transformation model in organ culture. In order to determine the levels of uptake of DMBA, the glands were exposed to the transforming dosage of the procarcinogen (7.8 microM, 20 microCi/ml) for 24 h, and the level of uptake was determined to be 8 x 10(4) cpm/mg of tissue. Subsequently, the glands were incubated in DMBA-free medium, and distribution of the radioactivity in DNA and in the acid-insoluble materials was measured. Data showed that, in addition to the 24-h DMBA treatment period, the initiation stage extends for another 3 h when the incubation is continued in DMBA-free medium. A saturation level of uptake of [3H]DMBA into the whole gland was observed at 12 h, while DMBA was continually metabolized with the products being bound to DNA and to the acid-insoluble fractions throughout the entire incubation period with or without DMBA. The three major adducts identified were anti-DMBA-3,4-diol-1,2-epoxide (DMBADE):deoxyguanosine, syn-DMBADE:deoxyadenosine, and anti-DMBADE:deoxyadenosine. Qualitatively the adduct profiles remained similar. However, with the additional 3-h incubation in DMBA-free medium, the three major DMBA-DNA adducts increased slightly by 6.5 to 7.5%. Thus the total 27-h time period can be considered as the duration of the initiation stage in the DMBA-induced carcinogenesis of mouse mammary cells in organ culture. Therefore the subsequent 6-h incubation in DMBA-free medium may be considered as within the promotional stage, and at the same time period the levels of the three DNA adducts decreased significantly by 67.5 to 84.1% (P less than 0.001).     

Circulating microRNA profiles reflect the presence of breast tumours but not the profiles of microRNAs within the tumours

Background

Extra-cellular microRNAs have been identified within blood and their profiles reflect various pathologies; therefore they have potential as disease biomarkers. Our aim was to investigate how circulating microRNA profiles change during cancer treatment. Our hypothesis was that tumour-related profiles are lost after tumour resection and therefore that comparison of profiles before and after surgery would allow identification of biomarker microRNAs. We aimed to examine whether these microRNAs were directly derived from tumours, and whether longitudinal expression monitoring could provide recurrence diagnoses.

Methods

Plasma was obtained from ten breast cancer patients before and at two time-points after resection. Tumour tissue was also obtained. Quantitative PCR were used to determine levels of 367 miRNAs. Relative expressions were determined after normalisation to miR-16, as is typical in the field, or to the mean microRNA level.

Results

210 microRNAs were detected in at least one plasma sample. Using miR-16 normalisation, we found few consistent changes in circulating microRNAs after resection, and statistical analyses indicated that this normalisation was not justifiable. However, using data normalised to mean microRNA expression we found a significant bias for levels of individual circulating microRNAs to be reduced after resection. Potential biomarker microRNAs were identified, including let-7b, let-7g and miR-18b, with higher levels associated with tumours. These microRNAs were over-represented within the more highly expressed microRNAs in matched tumours, suggesting that circulating populations are tumour-derived in part. Longitudinal monitoring did not allow early recurrence detection.

Conclusions

We concluded that specific circulating microRNAs may act as breast cancer biomarkers but methodological issues are critical.     

Long-term exposure to elevated levels of circulating TIMP-1 but not mammary TIMP-1 suppresses growth of mammary carcinomas in transgenic mice

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates matrix metalloproteinase activity, acts as a growth stimulator and inhibits apoptosis. We developed transgenic mice to evaluate the relevance of circulating versus mammary TIMP-1 in mammary carcinogenesis. The transgene was placed under the control of the albumin (Alb) promoter for the production of large amounts of TIMP-1 in the liver and release into the systemic circulation to achieve chronically elevated blood levels. The initial 7,12-dimethylbenz[a]anthracene (DMBA) mammary carcinogenesis study showed greatly decreased tumor incidence in heterozygous Alb-TIMP-1 mice (25%), compared with their wild-type (wt) littermates (83.3%). Metastatic mammary carcinomas were induced in the Alb-TIMP-1 mice through breeding with mice expressing the polyomavirus Middle T antigen (MT) under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Both the mammary tumor burden and the incidence of lung metastases were lower in the Alb-TIMP-1/MMTV-MT mice than their MMTV-MT littermates. Analysis of the Alb-TIMP-1/MMTV-MT tumors showed evidence of decreased proliferative activity and inhibition of apoptosis, whereas microvascular density was not affected. Transgenic expression of TIMP-1 in mammary epithelial cells was accomplished by using MMTV-LTR. In contrast to the Alb-TIMP-1 mice, there was insignificant difference in the growth of both DMBA- and MT-induced mammary tumors between heterozygous MMTV-TIMP-1 mice and their wt littermates. The MT-induced mammary tumors of the MMTV-TIMP-1 mice were separated into 'low' and 'high' TIMP-1 expressing groups. The 'high' TIMP-1 expressing tumors exhibited significantly higher proliferative activity than the tumors of the MMTV-MT only mice, whereas the number of apoptotic cells and microvascular density were not different. The findings of this study show that circulating TIMP-1, but not mammary-derived TIMP-1, has growth suppressive effects on DMBA and MT-induced mammary carcinomas.     

Enhancing effects of beta-estradiol 3-benzoate but not methoxychlor on the promotion/progression stage of chemically-induced mammary carcinogenesis in ovariectomized rats.

Modifying effects of beta-estradiol 3-benzoate (EB) and methoxychlor (MXC), a pesticide which possesses weak estrogenic activity, on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis were investigated in ovariectomized or intact female Sprague-Dawley rats. Twenty-eight weeks after a single DMBA (100 mg / kg body weight) initiation, when the incidence of mammary tumor-bearing rats had reached 75%, a number of the animals were subjected to ovariectomy in order to obtain 3 groups: i) tumor-bearing, ovariectomized group; ii) tumor-bearing, intact group; iii) no-tumor, ovariectomized group. Subsequently animals of each group were subjected to subcutaneous implantation of 0.5 mg EB or given diet containing 1000 ppm MXC for 13 weeks. Although the incidences, multiplicities and volumes of the palpable tumors gradually decreased after ovariectomy, EB treatment stimulated tumor growth in the tumor-bearing, ovariectomized group thereafter. A similar effect of EB treatment was also observed in the no-tumor, ovariectomized group. However, MXC did not show any effect in the tumor-bearing, or no-tumor ovariectomized groups, except that the multiplicity of tumors was significantly decreased by MXC treatment in the tumor-bearing, intact group. The results of our study suggest that MXC has no promotion / progression effect, but rather possesses a weak inhibitory effect, whereas the strongly estrogenic substance EB clearly enhanced DMBA-induced mammary tumorigenesis.     

Inducible NO synthase inhibits the growth of free tumor cells, but enhances the growth of solid tumors

To elucidate the role of nitric oxide (NO) in tumor cell growthin vivo, dynamic aspects of the growth of Ehrlich ascites tumorcells (EATCs) were studied in wild-type (WT) mice and in aninducible strain of NO synthase (iNOS)-deficient (iNOS^–/–)mice. Kinetic analysis showed that the rate of free tumor cellgrowth in the peritoneal cavity was significantly higher inthe iNOS^–/– mice than in the WT mice. In contrast,EATCs inoculated subcutaneously rapidly grew and formed a solidtumor in WT mice, but failed to grow in iNOS^–/–mice. These results clearly indicate that NO generated by iNOSpredominantly inhibits the growth of tumor cells in their freeform, but enhances the growth of solid tumors.     

Hydroxyurea enhances methylnitrosourea skin tumorigenesis when given shortly before, but not after, the carcinogen

To study the relationship between epidermal DNA synthesis andcarcinogenesis, hairless mice of both sexes were given a singletopical application of 1 mg N-methyl-N-nitrosourea (MNU) inacetone. A control group received only MNU, whereas other groupswere injected i.p. with 5 mg hydroxy-urea (HU), 1 h, 45 minand 15 min before, simultaneously with, and 15 min, 30 min and45 min after MNU application. The production of skin tumorswas recorded and the results were assessed with accepted statisticalmethods. Injection of HU shortly before a single applicationof MNU enhanced skin carcinogenesis, and when HU is injected30 min before MNU, the enhancement seems to be most pronounced.HU administered simultaneously with or following MNU application,did not alter the production of tumors. The cell kinetic situationin the epidermis at the time of a carcinogen application, andthe modulation of the cell kinetic reaction to the carcinogenby any type of post- or pretreatment, may influence tumorigenesis.     

In vitro growth promotion of human mammary carcinoma cells by steroid hormones, tamoxifen, and prolactin

Individually different growth responses of 10 cell lines newly derived from metastasizing mammary carcinomas were determined by cell counts in experimental incubations with the steroid hormones 17 beta-estradiol, progesterone, testosterone, hydrocortisone (cortisol), the antiestrogenic compound tamoxifen, or prolactin. Of 7 cell lines derived from ductal carcinomas, 5 were stimulated by prolactin. The growth of 4 of 7 cell lines established from the tumors of postmenopausal or ovariectomized patients was enhanced by doses of testosterone, which are in the range of the physiologic serum level. The proliferation of 5 cell lines was promoted by hydrocortisone in the physiologic concentration of 100 nM, supporting the notion that concentrations of testosterone or hydrocortisone normally present in body fluids may facilitate the in vivo growth of breast cancer. The in vitro growth of cells derived from tumors after relapse under treatment with medroxyprogesterone acetate or tamoxifen was markedly enhanced by progesterone or tamoxifen (CAS: 10540-29-1) in concentrations corresponding to therapeutical serum levels and in accordance with in vivo resistance to the endocrine therapy applied before cell sampling. The results of this suggest the occurrence of positive endocrine selection mechanisms operating in vivo on human mammary tumor cell populations.     

Morphology, growth characteristics and oestrogen-binding capacity of DMBA-induced mammary tumours from ovariectomized rats.

The morphology of 20 mammary adenocarcinomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) in Sprague-Dawley rats was compared with their growth characteristics and oestrogen-binding capacity following ovariectomy. The capacity to bind (3H)oestradiol-17B did not appear to be related to the growth characteristics, time of appearance after DMBA administration, or time between ovariectomy and assay for specific oestrogen-binding proteins. Furthermore, different tumours appeared to have oestrogen-binding capacities unrelated to the percentage of neoplastic cells within the tumour, amount of inflammation, mast cell infiltration, or the presence of fluid-filled cysts. The only morphological features which appeared to be correlated with oestrogen-binding capacity were the number of mitoses and the lipid content of the tumour; that is, the oestrogen-binding capacity tended to be lower in tumours with moderate or large numbers of mitoses and in tumours with much lipid in the epithelial cells. Six of the 19 adenocarcinomas found prior to sacrifice either continued growing or remained static following ovariectomy, while the others underwent regression. In 5 of the regressing tumours a new growth phase was observed, usually beginning 2 months after ovariectomy. Tumours other thus osteosarcoma as well as fibroadenomas and Zymbal-gland tumours.