N-nitrosomethylurea Research Articles

Histological analysis of low dose NMU effects in the rat mammary gland

BackgroundOur objective was to assess the histological changes in mammary glands of the female Wistar-Furth rat as a result of low dose exposure to N-nitrosomethylurea (NMU).MethodsGroups of 30–40 virgin female rats of between 49–58 days old received a single injection of 10, 20, 30 or 50 mg NMU/kg body weight (BW). A group of 10 control rats received 0.9% NaCl solution only. The formation of palpable mammary gland tumors was assessed weekly and, upon sacrifice at 12, 22 and 25–30 weeks after treatment, we performed a comprehensive histological analysis of all mammary gland lesions and tumors.ResultsAlongside the predicted increase in tumor number and decrease in tumor latency with increasing NMU dose, we observed a number of microscopic lesions and other epithelial abnormalities in the mammary glands for all NMU doses. Two types of non-neoplastic histological changes were observed in rats exposed to 10 or 20 mg NMU/kg BW: namely, (i) an increase in the number of acinar structures often accompanied by secretion into the lumen which is normally associated with pregnancy and lactation, and (ii) an increase in the number of epithelial cells sloughed into the lumen of the epithelial ducts.ConclusionThis study establishes a baseline for low-dose exposure and defines the histological features in the mammary gland resulting from NMU exposure. Furthermore, this system provides an ideal platform for evaluating the relative susceptibility of animals protected from, or predisposed to, developing cancer through environmental influences.

Citrus auraptene suppresses cyclin D1 and significantly delays N-methyl nitrosourea induced mammary carcinogenesis in female Sprague-Dawley rats

BackgroundBreast cancer is a major problem in the United States leading to tens of thousands of deaths each year. Although citrus auraptene suppresses cancer in numerous rodent models, its role in breast cancer prevention previously has not been reported. Thus, our goal was to determine the anticarcinogenic effects of auraptene against breast cancer.MethodsThe effects of auraptene on cell proliferation of MCF-7 and MDA-MB-231 human breast carcinoma cells in culture was assessed by measuring metabolism of a substrate to a formazan dye. Dietary effects of auraptene on tumor incidence, multiplicity and latency were studied in the N-methyl nitrosourea (MNU) induced mammary carcinogenesis model in female Sprague Dawley rats. The concentration of auraptene in rat tissues was analyzed by reverse phase HPLC. Cyclin D1 expression in MCF-7 cells and rat tumors was measured by western blot.ResultsAuraptene (500 ppm) significantly delayed median time to tumor by 39 days compared to the MNU only group (p < 0.05, n = 24–26). Auraptene (10 μM) reduced Insulin like Growth Factor-1 (IGF-1, 10 ng/mL)-induced cyclin D1 expression by 40% in MCF-7 cells. In comparison, western blot analysis of rat mammary tumors (n = 10 per group) confirmed that auraptene (500 ppm) significantly reduced (p < 0.05) cyclin D1 expression by 49% compared to the MNU only group. Analysis of rat mammary tissue extract by HPLC with fluorescence detection indicated an average concentration (means ± S.E.) of 1.4 ± 0.5 μM and 1.8 ± 0.3 μM in the normal mammary glands of the auraptene 200 ppm and 500 ppm groups, respectively. The concentration (means ± S.E.) of auraptene in the mammary tumors of the auraptene 200 ppm group was 0.31 ± 0.98 μM.ConclusionOverall, these observations suggest that the predominant effect of auraptene was to delay the development of tumors possibly through the suppression of cyclin D1 expression. These results point to the potential chemopreventive effects of auraptene in mammary carcinogenesis.

Diabetogenic effects of N-nitrosomethylurea in rats.

The influence of a single i.v. injection of 75 or 175 mg N-nitrosomethylurea (NMU)/kg into Wistar rats on plasma glucose, i.p. glucose tolerance, plasma insulin and glucagon, glucose stimulated insulin secretion and release as well as on the biosynthesis of insulin in isolated pancreatic islets have been investigated at set intervals between the 3rd day and the 6th week post injectionem. At the end of the experimental period all NMU-treated animals were alive but had significantly reduced body mass. All animals remained normoglycemic even after the glucose load. 3 days after drug application plasma insulin decreased only insignificantly in the 175 mg NMU-group, whereas plasma glucagon was significantly reduced in both drug-groups; after 3 and 6 weeks the reduction of plasma glucagon was still about 30 percent. In isolated islets the glucose stimulated insulin secretion, the percent stimulation of insulin release and insulin biosynthesis were significantly decreased.

Renin-Angiotensin System-Regulating Aminopeptidase Activities Are Modified in the Pineal Gland of Rats with Breast Cancer Induced by N-Methyl-Nitrosourea

Objective: Pineal function has been considered particularly as a neuroendocrine modulator in hormone responsive tumors, like the hormone-dependent mammary tumors. The complexity of the gland function, moreover, is denoted by the presence of a local renin-angiotensin-system (RAS) that regulates melatonin biosynthesis. Classically, angiotensin II (Ang II) has been considered as the effector peptide of the RAS, but Ang II is not the only active peptide. Several of its degradation products, including angiotensin III (Ang III) and angiotensin IV (Ang IV) also possess biological functions. These peptides are formed via the activity of several aminopeptidases. Our aim is to know their role in the regulation of pineal RAS and breast cancer. Design: Aminopeptidase N (APN), aminopeptidase B (APB) and aminopeptidase A (aspartyl- and glutamyl-aminopeptidase, APA) activities are measured in the pineal gland of rats with breast cancer induced by N-methyl nitrosourea (NMU). Methods: Aminopeptidase activities were measured fluorimetrically using their corresponding aminoacyl-ß-naphthylamides as substrates. Results: Specific APN and APB activities in pineal gland of controls and NMU-treated rats were not modified. Aspartyl aminopeptidase activity significantly decreased in NMU-treated rats when compared with control group. On the contrary, glutamyl aminopeptidase activity did not show significant differences between groups. Conclusions: We propose that the local RAS in pineal gland is modified in rats with breast cancer induced by NMU through the inhibition of AspAP activity, which may lead to increased levels of Ang II. Ang II could be responsible of the overproduction of melatonin, supporting a mechanism to restrain the promotion and/or progression of breast cancer.

Chemoprevention of MNU and Testosterone induced prostate carcinogenesis by Calcitriol (vitamin D3) in adult male albino Wistar rats

Background Calcitriol is a steroid hormone, inhibits the proliferation and promotes the differentiation of human prostate cancer cells. Calcitriol markedly inhibits the invasiveness of human prostate cancer cells in vitro. These properties support the use of calcitriol as differentiation therapy in prostate cancer. Chemopreventive role of calcitriol on prostate cancer remains unknown. Prostatic intraepithelial neoplasia (PIN) is the most common precancerous state and represents the major target for chemoprevention of prostate cancer.Methods Prostate cancer was induced in Wistar rats using MNU+T (N-Methyl nitroso urea + Testosterone). Calcitriol (0.5 μg/kg body weight) was administered weekly thrice as i.p. injection simultaneously to MNU+T treated rats. The control group received vehicle alone. After 16 weeks of experimental period ventral and dorsolateral lobes were removed for histopathological evaluation and serum prostatic acid phosphatase (PAcP) was determined.Results MNU+T treated rats showed hyperplasia, dysplasia and PIN (70%, 60% and 30%) changes in dorsolateral prostate and ventral prostate 60% 50% 30%, respectively. Where as MNU+T along with calcitriol treated rats, the incidence of hyperplasia, dysplasia and PIN in the ventral was 10% each and in dorsolateral it was 20%, 10% and 10%, respectively. Hyperplasia, dysplasia and PIN were less common in these rats. Serum PAcP significantly increases in MNU+T treated rats, whereas decreased in the calcitriol treated rats. The results of this study suggests that calcitriol may have chemopreventive activity in rat prostate carcinogenesis.Interpretation During the treatment with calcitriol on MNU+T induced prostate carcinogenesis, calcitriol might be capable of inhibiting the initiation of prostate cancer. Hence, calcitriol may have useful for the prevention of prostate cancer.

Global Gene Expression Profiling of Chemically Induced Rat Mammary Gland Carcinomas and Adenomas

Chemical carcinogens induce both benign and malignant mammary gland tumors in female Sprague-Dawley rats. To identify gene expression profiles associated with malignancy, cDNA microarray analysis was used to compare gene expression profiles in rat mammary gland carcinomas, adenomas, and normal mammary gland. Tumors were induced with various chemical carcinogens including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 7-12-dimethylbenz[a]anthracene (DMBA), N-nitrosomethylurea (NMU), and 4-aminobiphenyl. The global gene expression profiles in carcinomas and adenomas were distinguishable by hierarchical clustering and multi-dimensional scaling analyses. Permutation analysis revealed 110 clones statistically differentially expressed between benign and malignant tumors (p < 0.0005). Carcinomas showed relatively high expression of several genes associated with mammary epithelial cell growth and proliferation (e.g., cyclin D1, PDGFalpha) and relatively low expression of differentiation marker genes (e.g., beta -casein, whey acidic protein, transferrin). Other categories of genes showing differential expression between carcinomas and adenomas were associated with protein homeostasis, cytoskeleton, extracellular matrix, and cell metabolism (fatty acid metabolism, oxidative phosphorylation, and glycolysis). Major gene families implicated in malignancy by over-expression in carcinomas included the annexins (annexin A1 and A4) and Stat family of transcription factors (Stat3 and Stat5a). The elevated expression of the prolactin receptor in carcinomas concomitant with several components of the mitogenic prolactin signaling pathway implicated prolactin/prolactin receptor/Stat5a/cyclin D1 in rat mammary gland malignancy.

Endothelin B receptor agonist, IRL 1620, enhances the anti-tumor efficacy of paclitaxel in breast tumor rats

Pharmacological agents that increase tumor blood flow could be utilized to promote the delivery of anti-cancer drugs. We have demonstrated that administration of endothelin-1 (ET-1) to breast tumor bearing rats transiently increased tumor blood flow by stimulating endothelin B (ET(B)) receptors. The present study evaluated the effect of ET(B) receptor agonist, IRL 1620, on breast tumor perfusion, concentration of [3H]paclitaxel in tumor and tissues, and efficacy of paclitaxel in N-methyl nitrosourea induced breast tumor bearing rats. Administration of IRL 1620 (3 and 9 nmol/kg) significantly increased (203 and 140%, respectively) breast tumor perfusion. BQ 788, an ET(B) receptor antagonist, pretreatment completely abolished IRL 1620 induced increase in tumor perfusion. Tumor [3H]paclitaxel concentration was increased by 308% when [3H]paclitaxel was administered 15 min after IRL 1620 (3 nmol/kg) compared to vehicle treated rats. However, IRL 1620 did not increase [3H]paclitaxel concentrations in other organs. Efficacy study showed that paclitaxel (5 mg/kg) administration on every third day for a total of five doses produced 60.0, 4.5 and 0% reduction in tumor volume, tumor progression and complete tumor remission, respectively, compared to saline treated rats. However, paclitaxel (5 mg/kg) when administered 15 min after IRL 1620 (3 nmol/kg) produced 268.9, 210.3 and 20% reduction in tumor volume, tumor progression and complete remission of tumors, respectively, compared to saline treated rats. In conclusion, IRL 1620 significantly enhanced delivery and effectiveness of paclitaxel in an animal model of breast cancer.

Pyrrolidon Carboxypeptidase Activities in the Hypothalamus-pituitary-thyroid and Hypothalamus-pituitary-ovary Axes of Rats with Mammary Gland Cancer Induced by N-methyl Nitrosourea

Pyrrolidon carboxypeptidase is an omega-peptidase that hydrolyses N-terminal pyroglutamyl residues from biologically active peptides such as gonadotropin-releasing and thyrotrophin-releasing hormones. We previously described a decrease in both rat and human pyrrolidon carboxypeptidase activity with breast cancer, suggesting that gonadotropin-releasing hormone may be an important local intracrine, autocrine and/or paracrine hormonal factor in the pathogenesis of breast cancer while playing a role in the tumoral process. However, the other susceptible substrate of pyrrolidon carboxypeptidase, thyrotrophin-releasing hormone, may also be modified with breast cancer, supporting an association between breast cancer and thyroid disorders. The present work analyses soluble and membrane-bound pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes in N-methyl nitrosourea-induced breast cancer in rats. Our aim was to determine the possible relationship between gonadotropin-releasing hormone and thyrotrophin-releasing hormone regulation through pyrrolidon carboxypeptidase activity. We propose that pyrrolidon carboxypeptidase activity dysregulation at various local and systemic levels may participate in the initiation, promotion and progression of breast cancer induced in rat by N-methyl nitrosourea through the increase in gonadotropin-releasing hormone. Since pyrrolidon carboxypeptidase activity also acts on thyrotrophin-releasing hormone, the dysregulation of this enzyme's activity could indirectly affect hypothalamus-pituitary-thyroid axis function, and thus potentially represent a link between the diseases of thyroid and breast cancer.

Inhibition of NMU-induced mammary tumorigenesis by dietary soy

We previously demonstrated that female Sprague–Dawley rats fed AIN-93G diets containing soy protein isolate (SPI +) had lower DMBA-induced mammary tumor incidence than those fed diets containing casein (CAS), due partly to altered Phase I metabolism with soy. Here, we evaluated the tumor protective effects of these same diets to the direct-acting carcinogen N-methyl-nitrosourea (NMU). Tumor incidence was reduced and tumor latency was enhanced, in NMU-administered female rats lifetime exposed to SPI +, relative to the CAS group. Tumor multiplicity did not differ with diet, while tumor grade tended to be more advanced with SPI +. Normal mammary glands of CAS and SPI + tumor-bearing rats had comparable proliferative and apoptotic status. However, mammary expression of HER-2/neu and progesterone receptor (PR) genes was higher for SPI + rats. Moreover, tumored SPI + rats had lower serum progesterone levels than those fed CAS, while serum estrogen did not differ. Serum from tumored SPI + rats had higher apoptotic activity towards mammary epithelial MCF-7 cells, than CAS serum. Thus, dietary soy protects against mammary tumorigenesis induced by a direct-acting carcinogen and alters signaling pathways involving PR and HER-2/neu.

Specific enkephalin-degrading aminopeptidase activity in the HPT and HPO axes of rats with breast cancer induced by N-methyl nitrosourea

State and function of breast depend on an endocrinological balance, the upsetting of which can be a factor favorable to the development of cancer. Enkephalins (ENK) have been considered as a particular form of adaptation to defense to the organism against neoplastic processes. However, ENK may modify the endocrine functions of glands such as the ovary or the thyroid through the hypothalamus–pituitary axis, acting direct or indirectly as endocrine, paracrine or autocrine stimulatory growth factors. The present work analyses enkephalin-degrading tyrosyl aminopeptidase (EDA) activity in the hypothalamus–pituitary–thyroid (HPT) and hypothalamus–pituitary–ovary (HPO) axes in a rat model of breast cancer induced by N-methyl-nitrosourea (NMU) to state the relationship between ENK levels modification through EDA activity at different neuroendocrine levels and breast cancer. Results obtained show a decrease in EDA activity in hypothalamus, anterior and posterior pituitary, thyroid and ovary, suggesting increased levels of ENK in all these locations. These ENK may induce breast cancer cell growth and progression not only at breast level, but also acting at several neuroendocrine levels such as the HPT and HPO axes, inducing an unbalance of several other hormones, which could also facilitate the progression of cancer as an undesirable concomitant effect.

Serum oxytocinase activity is related to tumor growth parameters in N-methyl nitrosourea induced rat breast cancer

Oxytocinase has been reported to hydrolyse the peptide hormone oxytocin (OT). We have previously described changes in oxytocinase activity in human breast cancer, where a highly significant increase occurred in tumoral tissue. In the present work, we analysed oxytocinase activity in serum of rats with breast cancer induced by N-methyl-nitrosourea (NMU). We also correlated these data with the number and size of tumors and the body weight of the animals to evaluate the putative value of this activity as a biological marker of the disease. Our results confirm the involvement of OT in carcinogenesis and suggest a mayor role for oxytocinase activity in the development of breast cancer.

The stroma as a crucial target in rat mammary gland carcinogenesis.

A complex network of interactions between the stroma, the extracellular matrix and the epithelium drives mammary gland development and function. Two main assumptions in chemical carcinogenesis of the mammary gland have been that carcinogens induce neoplasia by causing mutations in the DNA of the epithelial cells and that the alterations of tissue architecture observed in neoplasms are a consequence of this primary mutational event. Here, we use a rat mammary tissue recombination model and the chemical carcinogen N-nitrosomethylurea (NMU) to determine whether the primary target of the carcinogen is the epithelium, the stroma or both tissue compartments. Mammary epithelial cells were exposed in vitro either to the carcinogen or vehicle before being transplanted into the cleared fat pads of rats exposed to carcinogen or vehicle. We observed that neoplastic transformation of these mammary epithelial cells occurred only when the stroma was exposed in vivo to NMU, regardless of whether or not the epithelial cells were exposed to the carcinogen. Mammary epithelial cells exposed in vitro to the carcinogen formed phenotypically normal ducts when injected into a non-treated stroma. Mutation in the Ha-ras-1 gene did not correlate with initiation of neoplasia. Not only was it often found in both cleared mammary fat pads of vehicle-treated animals and intact mammary glands of untreated animals, but it was also absent in some tumors. Our results suggest that the stroma is a crucial target of the carcinogen and that mutation in the Ha-ras-1 gene is neither necessary nor sufficient for tumor initiation.

Enhanced radiosensitivity of rat autochthonous mammary tumors by dietary docosahexaenoic acid.

Dietary docosahexaenoic acid (DHA), which integrates into tumor cell membranes, has been reported to enhance the efficacy against tumors of cytotoxic drugs that induce reactive oxygen species (ROS). Because ionizing radiation also generate ROS, we initiated a study to determine whether dietary DHA might sensitize mammary tumors to irradiation. Mammary tumors were induced by N-methylnitrosourea (NMU) in Sprague-Dawley rats. The optimal dose of radiation to examine the effect of DHA on tumor response to irradiation was determined to be 18 grays (Gy) using a 4-6 MeV electron beam (according to the depth of the target volume) delivered in a single fraction from a linear accelerator. Two groups of rats were fed a basal diet containing 7% of a mixture of peanut and rapeseed oils enriched with 8% of an oil containing either a low (palm oil) or high (DHASCO oil containing 40% DHA) DHA content. DHA group was equally subdivided into 2 groups without or with addition of vitamin E (100 IU/kg diet). Irradiation was carried out when the first tumor in each rat reached 1.5 cm2 and subsequent change in tumor size was documented over time. DHA level in adipose tissue, taken as a biomarker, was higher in the DHA supplemented group compared to the control group. Vitamin E level in liver, the best storage for this compound, was higher in the vitamin E supplemented DHA group compared to the DHA group. Tumor size decreased by 60% at 12 days after irradiation in the DHA group vs. 31% in the control group (p = 0.03) and 36% in the DHA plus vitamin E group. Therefore, dietary DHA sensitized mammary tumors to radiation. The addition of vitamin E inhibited the beneficial effect of DHA, suggesting that this effect might be mediated by oxidative damage to the peroxidizable lipids.

Serum Pyrrolidone Carboxypeptidase Activity in N-methyl Nitrosourea Induced Rat Breast Cancer

Pyrrolidone carboxypeptidase (Pcp) (E.C. 3.4.19.3) is an omega peptidase widely distributed in animal fluids and tissues and hydrolyses N-terminal pyroglutamic residues from biologically active peptides such as gonadotropin releasing hormone (GnRH). Previous results obtained by us showed a decrease in human breast cancer Pcp activity, suggesting that this enzyme activity or its putative substrates may play a major role in breast cancer pathogenesis. The aim of the present work is to analyse serum Pcp activity in N-methyl-nitrosourea (NMU) induced rat mammary tumours using pyroglutamyl-beta-naphthylamide as substrate. Serum Pcp activity was significantly lower in NMU-treated rats than in controls. Moreover, multiple regression analysis showed a significant correlation between Pcp activity and the number and size of tumours and the body weight of the animals. Since NMU-induced carcinomas are mainly oestrogen-dependent, the decrease observed in Pcp activity may reflect an increase in circulating levels of GnRH that lead to an increase in gonadal steroid hormones production responsible, at least in part, for the initiation and promotion of the disease.

VIP grafted sterically stabilized liposomes for targeted imaging of breast cancer: in vivo studies

Targeted delivery of radionuclides and therapeutic agents to specific biomarkers of breast cancer has important implications for the diagnosis and therapy of breast cancer. Vasoactive intestinal peptide receptors (VIP-R) are approximately five times more expressed in human breast cancer, compared to normal breast tissue. We have used VIP, a 28 amino acid mammalian neuropeptide, as a breast cancer targeting moiety for targeted imaging of breast cancer. VIP was covalently attached to the surface of sterically stabilized liposomes (SSL) that encapsulated a radionuclide, Tc99m-HMPAO. Rats with n-methyl nitrosourea (MNU)-induced in situ breast cancers were used to test this targeted liposomal imaging agent. Specifically, the pharmacokinetics and biodistribution of Tc99m-HMPAO encapsulating SSL with and without VIP were determined together with their ability to image breast cancer. The presence of VIP did not alter the size and Tc99m-HMPAO encapsulation ability of SSL. It also did not alter the pharmacokinetic profile of SSL. Long-circulating liposomes with and without VIP on their surface accumulated at significantly higher quantities in breast cancer when compared to normal breast, indicating passive targeting of these constructs to cancer tissues. Importantly, in breast cancer, Tc99m-HMPAO encapsulating SSL with VIP showed significantly more accumulation than SSL without VIP. The tumor to non-tumor ratio was also significantly higher for Tc99m-HMPAO encapsulating VIP–SSL than Tc99m-HMPAO encapsulating SSL without VIP, suggesting active targeting of VIP–SSL to breast cancer. Collectively, these data showed that Tc99m-HMPAO encapsulating VIP–SSL can be successfully used for the targeted imaging of breast cancer.

Preparation and biological evaluation of the new chlorin photosensitizer T3,4BCPC for detection and treatment of tumors

The new water-soluble photosensitizer 5,10,15,20-tetrakis[3,4-bis(carboxymethyleneoxy)phenyl]chlorin (T3,4BCPC) has been prepared, characterized and labeled with 99mTc radionuclide. The radiotracer was evaluated for tissue distribution in Wistar rats. Accumulation of administrated activities in the liver, kidney, bladder and large intestine at 4 h post-injection indicated that the labeled ligand was largely eliminated through the renal and partly through the hepatobiliary system. In vivo biodistribution studies of the labeled compound were carried out in rodent and murine tumor models in comparison with other tumor-seeking radiopharmaceuticals such as 99mTc(V)-dimercaptosuccinic acid (DMSA), 201thallous chloride (TlCl) and 99mTc-citrate using a gamma camera computer system. In N-nitrosomethylurea (NMU)-induced rat mammary tumors, the labeled ligand showed a five-fold tumor to muscle (T/M) ratio compared to 99mTc(V)-DMSA (3-fold) and 201TlCl (3-fold). In the case of C 3H/J virus-induced spontaneous mammary tumors, the differences were not marked. However, in the transplanted rat C 6-glioma, the T/M ratio of the labeled compound was appreciably higher (four-fold) than that noted with 99mTc(V)-DMSA (two-fold), 201TlCl (three-fold) and 99mTc-citrate (more than three-fold). These findings suggest that the radiolabeled T3,4BCPC may have potential for the detection of cancer. In order to ascertain the efficacy of the compound for photodynamic therapy applications, a preclinical PDT study was carried out in fibrosarcoma-bearing mice after injecting 5.0 mg/kg body weight of the T3,4BCPC. A laser dose of 20 mW for 60 s resulted in 80% destruction of tumors. These data suggest that this molecule could be useful for PDT of cancer. The labeled agent could also be useful in monitoring the progression/regression of tumors before, during, and after chemotherapy, radiation therapy or PDT.

Thymosin α 1 inhibits mammary carcinogenesis in Fisher rats

The effects of thymosin α 1 (Tα 1) on mammary carcinogenesis was investigated in Fisher rats. Mammary carcinomas were observed 3 months after N-nitrosomethylurea (NMU) injection (10 mg, i.p.) into Fisher rats. Daily administration of Tα 1 (10 μg, s.c.) reduced mammary carcinoma incidence and prolonged survival time. Animals treated with exogenous Tα 1 had a significantly greater blood white cell density than control Fisher rats. These results suggest that Tα 1 prevents mammary carcinoma incidence as a result of stimulation of the immune system.

Dietary genistein suppresses chemically induced prostate cancer in Lobund–Wistar rats

Epidemiological reports suggest that Asians consuming a diet high in soy have a low incidence of clinically manifested prostate cancer. We have tested the hypothesis that life-time exposure to genistein, the primary isoflavone component of soy, is responsible for this protective effect. Lobund–Wistar rats were exposed to 0, 25 and 250 mg genistein/kg AIN-76A diet, starting at conception and continued until necropsy at 11 months. Male offspring were injected s.c. with Flutamide on days 50-66 and with testosterone on days 67-69, injected with N-methylnitrosourea (NMU) into the dorsal prostate on day 70, and given testosterone implants, starting at day 77. Genistein in the diet inhibited the development of NMU-induced prostate invasive adenocarcinomas, in a dose-dependent manner. Genistein did not alter body, prostate and testes weights. Male rats fed 0, 25 and 250 mg genistein/kg diet had serum genistein concentrations of 9, 60 and 861 pmol/ml, and prostate genistein concentrations of 85, 230 and 775 pmol/g tissue. We conclude that lifetime dietary genistein protected against chemically induced prostate cancer development in rats.

VIP receptors as molecular targets of breast cancer: implications for targeted imaging and drug delivery

Receptors for vasoactive intestinal peptide (VIP-R) are overexpressed in human breast cancer. This phenomenon may have important diagnostic and therapeutic implications because carrier systems loaded with imaging or therapeutic agents, and with surface ligands to VIP-R could potentially be actively targeted to breast cancer. Previously, we have prepared sterically stabilized liposomes (SSL) with VIP non-covalently associated on their surface. However, these liposomes were not able to actively target to breast cancer in rats in situ, most probably due to dissociation of non-covalently associated VIP from SSL. Hence, there is a need to conjugate VIP covalently to SSL. This study aims to begin to address this issue and to test the targeting ability of VIP–SSL to n-methyl nitrosourea (MNU)-induced rat breast cancer in vitro. First, VIP was conjugated to DSPE–PEG 3400–NHS [1,2-dioleoyl- sn-glycero-3-phosphoethanolamine- n-[poly(ethylene glycol)]- N-hydroxy succinamide, PEG M w 3400] under mild conditions to obtain a predominantly 1:1 conjugate of VIP and DSPE–PEG 3400 (DSPE–PEG 3400–VIP), as evidenced by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Next, DSPE–PEG 3400–VIP was inserted into preformed fluorescent cholesterol (BODIPY-Chol) labeled SSL by incubation at 37°C. To test breast cancer targeting ability in vitro, these VIP–SSL were subsequently incubated with MNU-induced rat breast cancer tissue sections. The results showed that when compared to fluorescent SSL without VIP or non-covalently attached VIP, significantly more VIP–SSL were attached to rat breast cancer tissues indicating that SSL with covalently attached VIP can be actively targeted to rat breast cancer tissues. This targeted carrier system is currently being explored for functional imaging and targeted chemotherapy of breast cancer.

EFFECTS OF VITAMIN D (CALCITRIOL) ON TRANSITIONAL CELL CARCINOMA OF THE BLADDER IN VITRO AND IN VIVO

Vitamin D (calcitriol) has significant antiproliferative effects on various tumor cells in vitro and in vivo. In the clinical situation a major impediment to systemic administration of calcitriol is the side effect of hypercalcemia. To test the potential usefulness of calcitriol for bladder cancer treatment, we studied the antiproliferative effect of vitamin D on 2 human bladder cancer cell lines, 253j and T-24, in vitro. We also examined the in vivo effects of calcitriol in an animal model of bladder cancer using intravesical administration to avoid the toxicity of systemic calcitriol therapy. The presence of vitamin D receptors in normal and neoplastic human bladder tissue, and tumor cells T-24 and 253j was determined by immunoblot analysis. Tumor cell proliferation in the presence or absence of calcitriol was determined using a crystal violet assay. Calcitriol induced apoptosis was determined by morphology, polyadenosine diphosphate ribose polymerase cleavage and annexin V binding. In vivo studies were performed by weekly intravesical instillation of calcitriol in female Fischer 344 rats after induction of tumors by N-methyl nitrosourea. Calcitriol administration was started 3 weeks after tumor induction for 7 doses at weekly intervals. Normal and neoplastic human bladder tissue, and the cell lines expressed vitamin D receptors. In the 253j and T-24 cell lines proliferation was significantly inhibited by calcitriol. Progressive cleavage of full length polyadenosine diphosphate ribose polymerase was observed in calcitriol treated cells starting as early as 4 hours after exposure. Similar changes were not observed in the control cells treated with vehicle (ethanol) alone. After 24 hours of treatment with calcitriol 45.8% of 253j cells bound annexin compared to 16.5% of control cells (chi-square p <0.001). Of the control animals 66% developed bladder tumors and 55% of the animals treated with calcitriol early (3 weeks) after tumor induction developed bladder tumors. Almost all of the tumors that developed in the calcitriol group were unifocal, and only 20% were invasive compared to 50% of those in the control animals. These results demonstrate that calcitriol inhibits proliferation and induces apoptosis in human bladder tumor cells in vitro, and may have therapeutic potential in bladder cancer. In vivo studies using an N-methylnitrosourea induced model of bladder cancer demonstrate that early institution of intravesical calcitriol therapy after carcinogen exposure results in fewer tumors, which are also less likely to be multifocal, high grade or invasive. With our protocol a short course of intravesical calcitriol administration did not result in any significant toxicity.