Rat Mammary Tumor Model Research Articles

PPARβ mRNA expression, reduced by n − 3 PUFA diet in mammary tumor, controls breast cancer cell growth

The effect of numerous anticancer drugs on breast cancer cell lines and rodent mammary tumors can be enhanced by a treatment with long-chain n−3 polyunsaturated fatty acids (n−3 PUFA) such as docosahexaenoic acid (DHA, 22:6n−3) which is a natural ligand of peroxisome proliferator-activated receptors (PPAR). In order to identify the PPAR regulating breast cancer cell growth, we tested the impact of siRNA, selected to suppress PPARα, PPARβ or PPARγ mRNA in MDA-MB-231 and MCF-7 breast cancer cell lines. The siPPARβ was the most effective to inhibit breast cancer cell growth in both cell lines. Using PPARα, PPARβ and PPARγ pharmacological antagonists, we showed that PPARβ regulated DHA-induced inhibition of growth in MDA-MB-231 and MCF-7 cells. In addition, the expressions of all 3 PPAR mRNA were co-regulated in both cell lines, upon treatments with siRNA or PPAR antagonists. PPAR mRNA expression was also examined in the NitrosoMethylUrea (NMU)-induced rat mammary tumor model. The expressions of PPARα and PPARβ mRNAs were correlated in the control group but not in the n−3 PUFA group in which the expression of PPARβ mRNA was reduced. Although PPARα expression was also increased in the n−3 PUFA-enriched diet group under docetaxel treatment, it is only the expression of PPARβ mRNA that correlated with the regression of mammary tumors: those that most regressed displayed the lowest PPARβ mRNA expression. Altogether, these data identify PPARβ as an important player capable of modulating other PPAR mRNA expressions, under DHA diet, for inhibiting breast cancer cell growth and mammary tumor growth.

Dietary modulation of BALT on a rat mammary tumor model

Murine bronchus associated lymphoid tissue (BALT) is a mirror of systemic immunity since it varies greatly with immune status of the animal and plays a pivotal role in local immune responses at the lung surface. BALT enhanced histopathology (EH) is a semiquantitative screening tool to determine whether there is a potential effect on the immune system of rats under controlled conditions.168 Sprague Dawley virgin female rats with 42 days were randomly distributed for seven groups of 24 animals each. Besides group I, at 55 days the groups were induced for mammary cancer with 20mg of 1,12‐dimetilbenzantracene (DMBA). Dietary protocols were applied for 150 days as following. Group I: standard diet (SD), 300Kcal/100g; Group II: DMBA +SD; Group III: SD + beans 40g/Kg of SD, 341Kcal/100g; Group IV: SD + olive oil (50ml/Kg of SD); Group V: fiber supplement 4× the SD, 250Kcal/100g; Group VI: fiber supplement 4× the SD + beans 19,95gr + olive oil 25ml on 1Kg of SD; Group VII: SD + mediterranean lyophilized diet (41Kcal). The animals were ethically euthanized and the lungs were routinely fixed and stained with hematoxylin‐eosin. BALT was evaluated by EH.The BALT morphology of tumoral rats with mediterranean diet and caloric restrain was similar to the BALT of control group. The other tumoral groups had decreased BALT in size and lymphocyte number compared to control group.These results suggest that diet can interfere on BALT morphology.

Inhibition of Estrogen-Mediated Mammary Tumorigenesis by Blueberry and Black Raspberry

We previously demonstrated the protective effects of blueberry (BB) and black raspberry (BRB) supplemented at 2.5% dose in an ACI rat mammary tumor model. Here, we assessed a dose-related alteration in tumor indices with diet supplemented with 5% BB or BRB powder. The diet was well tolerated. Tumor palpation from 12 weeks revealed first tumor appearance by 84 days in the control group, that was delayed by 24 and 39 days with the BB and BRB diets, respectively (p = 0.04). Ellagic acid detected in the plasma of rats fed the BRB diet was in the range of 96.6-294.2 ng/mL. While the BB diet showed better efficacy in reducing mammary tissue proliferation and tumor burden, tumor latency was delayed efficiently by BRB. Furthermore, BB was effective in downregulating CYP1A1 expression, while BRB downregulated ERα expression effectively. Distinct anticarcinogenic effects of the two berries correspond to their distinct phytochemical signatures.

Anti-tumor effects of a novel retinoic acid metabolism blocking agent VN/14-1 in the N-methyl-N-nitrosourea-induced rat mammary carcinoma model and its effects on the uterus

VN/14-1 [4-(±)-(1H-Imidazol-1-yl)-(E)-retinoic acid], a novel retinoic acid metabolism blocking agent (RAMBA), works by inhibiting the breakdown of all-trans-retinoic acid. The purpose of this study was to evaluate the anti-tumor effects of VN/14-1 on the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma model, and peripheral organ effects on the uteri of immature ovariectomized (OVX) rats. In tumor burden experiments, after 56 days of administration of VN/14-1 5, 10, and 20 mg/kg/day, significant tumor reductions in mean tumor weight of 19.1, 34.4, and 44.3%, compared to tumors in control animals occurred. Cumulative tumor growth was also significantly slower in a dose-dependent manner in groups receiving 5, 10, and 20 mg/kg/day of VN/14-1 compared to growth rates in the control group. Tumor apoptosis was significant increases in animals treated with 5, 10, and 20 mg/kg/day of VN/14-1. In uterotrophic experiments, immature OVX rats given VN/14-1 significantly reduced uterine weight and blocked endometrial stimulation induced by unopposed β-estradiol (E2). In both rat models, adverse toxicities included weakness, anorexia, and reduction in body weight in the groups given the highest dose of 20 mg/kg/day. In summary, VN/14-1 inhibited tumor growth in the MNU-induced estrogen receptor (ER)-positive rat mammary tumor model, and antagonized the stimulatory effect of estrogens on the uterus. The studies suggest that VN/14-1 may be a useful novel therapy for ER-positive breast cancer.

Magnetomotive molecular nanoprobes.

Tremendous developments in the field of biomedical imaging in the past two decades have resulted in the transformation of anatomical imaging to molecular-specific imaging. The main approaches towards imaging at a molecular level are the development of high resolution imaging modalities with high penetration depths and increased sensitivity, and the development of molecular probes with high specificity. The development of novel molecular contrast agents and their success in molecular optical imaging modalities have lead to the emergence of molecular optical imaging as a more versatile and capable technique for providing morphological, spatial, and functional information at the molecular level with high sensitivity and precision, compared to other imaging modalities. In this review, we discuss a new class of dynamic contrast agents called magnetomotive molecular nanoprobes for molecular-specific imaging. Magnetomotive agents are superparamagnetic nanoparticles, typically iron-oxide, that are physically displaced by the application of a small modulating external magnetic field. Dynamic phase-sensitive position measurements are performed using any high resolution imaging modality, including optical coherence tomography (OCT), ultrasonography, or magnetic resonance imaging (MRI). The dynamics of the magnetomotive agents can be used to extract the biomechanical tissue properties in which the nanoparticles are bound, and the agents can be used to deliver therapy via magnetomotive displacements to modulate or disrupt cell function, or hyperthermia to kill cells. These agents can be targeted via conjugation to antibodies, and in vivo targeted imaging has been shown in a carcinogeninduced rat mammary tumor model. The iron-oxide nanoparticles also exhibit negative T2 contrast in MRI, and modulations can produce ultrasound imaging contrast for multimodal imaging applications.

Abstract 1450: Loss of the UPR member CREB3L1 is vital for the survival of breast cancer cells in the tumor microenvironment and development of the metastatic phenotype

Abstract The unfolded protein response (UPR) is required for proper protein folding in the endoplasmic reticulum under conditions of stress such as those encountered in the tumor microenvironment. Recently, a number of studies have suggested a role for the unfolded protein response in the development of cancer, more specifically in regulating the balance between cell death, dormancy and the growth of cancer cells in the tumor microenvironment. This study examined the role of the transcription factor CREB3L1, a member of the UPR machinery, in breast cancer development and metastasis. Northern blotting analysis identified a loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low- or non-metastatic cell lines, which was subsequently shown, through bisulphate sequencing, to be the result of enhanced promoter methylation. When metastatic cells were transfected with CREB3L1 they demonstrated a significant decrease in survival in hypoxic and non-adherent growth conditions, and reduced in vitro invasion and migration in chemotaxis assays. Interestingly, in an in vivo rat mammary tumor model where rats were injected with cells in the hind footpad, the CREB3L1 expressing rat cells not only failed to form metastases but tumor regression was seen in 70% of the animals. In contrast, metastases were seen in the popliteal lymph node of 90% of rats injected with wild-type and empty vector-transfected cells. Microarray and ChIP on Chip analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis when CREB3L1 was expressed. We also observed a significant decrease in the number of migrating endothelial cells in scratch and chemotaxis assays in media conditioned by CREB3L1 expressing cells, compared to conditioned media from untransfected cells. To determine if these findings translated to human breast cancer, real-time PCR analysis of CREB3L1 expression in human breast cancer tissues of differing grade (n=30) was performed. Expression of CREB3L1 was elevated in low and medium grade tissue but substantially reduced in high grade tissue, compared to normal. The high grade breast cancer tissues demonstrated a 6.6 fold decrease in the expression of CREB3L1 compared to the low grade tissues (p<0.001), with about a 2 fold decrease compared to normal tissue. In conclusion, CREB3L1 expression is elevated in low and medium grade breast cancer tissue, perhaps as the cancer cells respond to various stresses. However, loss of CREB3L1 expression is required for the long-term survival of breast cancer cells in the tumor microenvironment and the progression to a highly metastatic phenotype. CREB3L1 expression may be a useful marker in predicting tumor grade and patient prognosis, in addition to providing a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2011-1450

Discovery of LY2457546: a multi-targeted anti-angiogenic kinase inhibitor with a novel spectrum of activity and exquisite potency in the acute myelogenous leukemia-Flt-3-internal tandem duplication mutant human tumor xenograft model

LY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRβ, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non-clinical safety profile justify the advancement of LY2457546 into clinical testing.

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

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

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[18F]fluoro-glucose (FDG) and 3′-deoxy-3′-[18F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC50 values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 µM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.

Abstract 4766: Multivalent VRP-based anti-tumor immunotherapy is an effective therapeutic strategy in a rat model of breast cancer

Abstract It is widely accepted that the genetic instability of a tumor results in both intra- and inter-tumoral genetic heterogeneity, which leads to heterogeneous tumor-associated antigen (TAA) expression and contributes to the outgrowth of antigen-loss variants in response to immunologic selective pressure. Using a rat neu encoding, alphavirus based virus-like replicon particle (VRP) with restricted in vivo tropism for dendritic cells in a fully tolerant Fisher 344 rat mammary tumor (13762 MAT B III) model, we previously demonstrated the ability to cure a modest proportion, 20-40%, of rats with pre-existing mammary tumors and observed the outgrowth of antigen loss variant tumors in a proportion of animals that ultimately succumbed to their tumor. Thus, we hypothesized that strategies targeting multiple tumor antigens would lead to improved treatment efficacy. Expression of the cancer testes antigen, Brother of Regulator of Imprinted Sites (BORIS) was confirmed in the 13762 MAT B III tumor. Rats with pre-existing tumors were treated with VRP encoding either the TAA rat neu or the BORIS target antigen, alone or in combination. We found that targeting either rat neu or BORIS alone was effective in treating a proportion of animals. Interestingly, administration of both VRP agents at a single anatomic site drastically decreased the treatment efficacy, by approximately 50%, while separating their administration into two separate anatomic sites enhanced the treatment efficacy over that provided by each agent alone, to &amp;gt;56% cure rate. Herein, we will present data addressing potential mechanisms for these treatment observations. To our knowledge, this is the first report of multivalent anti-tumor immunotherapy utilizing solely the VRP vector system. These data represent an exceptionally promising platform upon which to build more efficacious immunotherapeutic strategies including those targeting multiple defined TAAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4766.

Induction of mammary gland tumor in female Sprague- Dawley rats with LA7 cells

The current methods for tumor induction in breast cancer research animal models are time-consuming, hazardous, expensive, sometimes irreproducible and inconvenient. We successfully developed a new, simple and cost-effective method in developing solid mammary gland tumor in female Sprague-Dawley rat using LA7 rat mammary tumor cells. Tumors developed in 7- 8 weeks old rats within 6 to 8 days of subcutaneous injection of LA7 cells into the mammary gland pad. Tumor size increased exponentially for four weeks. Histopathology examination confirmed that the induced tumors were adenocarcinomas. Evaluation of blood enzymes showed significantly higher (P < 0.005) serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in tumor-bearing than in normal rats. This LA7 cell-induced rat mammary gland tumor model may be useful for studies in breast cancer drug or nutraceutical research and development.

Intraductal pegylated liposomal doxirubicin may achieve long term protection in HER2/NEU transgenic mice by restricting mammary gland outgrowth

This work is based on the hypothesis that intraductal injection can treat existing lesions and prevent future breast cancers originating in the epithelial cells lining the breast ducts. We have previously demonstrated our ability to access the entire mammary gland through the teat in mouse and rat mammary tumor models. Further, we showed that several other common chemotherapeutic drugs such as 5 fluorouracil, carboplatin, and methotrexate were effective, but not to the same extent as PLD. Thus, intraductal injection of PLD has potential in the prevention and neo-adjuvant therapy of breast cancer. Recently, we observed that in addition to rendering the mammary glands tumor free for more than 3 months after all of the control animals had developed multiple tumors, mammary glands of PLD treated mice were stunted in their growth. Instead of the florid growth with multiple branches and side branches studded with terminal end buds, akin to a spring tree seen in normal glands the PLD treated mammary gland presented the aspect of an autumn tree. In the transgenic mice, the epithelial cells in the entire mammary gland express the MMTV-Her2/neu transgene and at even higher levels during pregnancy. This raised the concern that stimulation of proliferation of these cells, for example, by pregnancy, may result in a higher incidence of spontaneous mammary tumors in the PLD treated mice. What then is the response of this treated mammary gland to a new pregnancy? To test this concept, we induced pregnancy in PLD treated mice. Her2/neu mice treated with intraductal PLD, like their untreated controls, had normal deliveries, with pups of normal weight and number in the litter. However, unlike control mice, their pups survived for less than one week. Long term follow-up showed that pregnancy did not increase the incidence of tumors in PLD treated mice. Their mammary glands showed a very poor proliferative response to the pregnancy hormones and remained stunted in their growth. It is likely that PLD not only affected the eradication of preneoplasias but also resulted in a depletion of normal mammary gland stem cells This concept is being tested by determining the difference in stem cell content between untreated and PLD treated glands by transplantation of serial dilutions of epithelial cells from sham and PLD treated glands into cleared mouse fat pads, examination of markers of stem cells, such as ALDHA, etc. If proven to be the case, these findings raise the possibility that long term protection may be achieved by intraductal injection of PLD to women at high risk of developing breast cancer.

Chemoprevention of rat mammary carcinogenesis by Azadirachta indica leaf fractions: Modulation of hormone status, xenobiotic-metabolizing enzymes, oxidative stress, cell proliferation and apoptosis

We evaluated the chemopreventive potential of the ethyl acetate fraction (EAF) and methanolic fraction (MF) of Azadirachta indica (neem) leaf on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis. Estradiol and estrogen receptor status, xenobiotic-metabolizing enzyme activities, redox status, DNA and protein modifications, and the expression of cell proliferation, and apoptosis related proteins in the mammary gland and liver were used as biomarkers of chemoprevention. Administration of both EAF and MF at a dose of 10 mg/kg bw effectively suppressed tumour incidence. Chemoprevention by neem leaf fractions was associated with modulation of hormone and receptor status, xenobiotic-metabolising enzymes, and lipid and protein oxidation, with upregulation of antioxidants, inhibition of oxidative DNA damage, protein modification, and cell proliferation, and induction of apoptosis. However EAF rich in constituent phytochemicals was more effective than MF in modulating multiple molecular targets. These results provide evidence for the chemopreventive efficacy of neem leaf fractions in the rat mammary tumour model.

Optical coherence tomography (OCT) as a diagnostic tool for the real-time intraoperative assessment of breast cancer surgical margins.

Abstract Abstract #802 Background: The decrease in the number of breast cancer deaths has largely been attributed to increased awareness, earlier detection, and improved treatment options. However, as the number of breast-conserving surgeries rose over the years, the need for negative margins and little or no residual disease has become critical to help reduce the chances of local recurrence. OCT is a high resolution imaging modality that has been used to image tumor margins in an NMU-carcinogen-induced rat mammary tumor model. Due to the location of breast lesions, the use of needle-based imaging probes may be used to further extend the reach of the OCT imaging beam by incorporating an optical fiber into biopsy needle tips, providing real-time information to guide biopsies or to place localization wires.&amp;#x2028; Material &amp; Methods: A clinical spectral domain OCT system was developed with a super luminescent diode light source centered at 1310 nm with a bandwidth of 92 nm yielding an axial resolution of 8.3 µm. The beam delivery sample arm uses a 60 mm achromatic lens to focus 4.75 mW of light to a 35.0 µm spot size (transverse resolution) with a confocal parameter of 1.47 mm. The patients included in this study had primary breast tumors diagnosed by needle-biopsy and were in need of surgical resection, as determined by their physicians. At Carle Foundation Hospital, the OCT system was placed inside the operating room during breast conserving surgical procedures to image the tissue specimens. The OCT images were evaluated by a single operator allowing for consistent classification based on the level of scattering intensity and heterogeneity, scattering profile, and physical extent of the highly scattering area.&amp;#x2028; Results: An initial training data set of OCT images from 17 patients was used to establish standard imaging protocols and standard evaluation criteria of the surgical margins. Of the 20 additional tissue specimen imaged for the feasibility study, 11 were identified as having a positive or close surgical margin and nine as a negative margin under OCT. In comparing to the H&amp;E histology, there were 9 true positives, 9 true negatives, 2 false positives, and 0 false negatives yielding a sensitivity of 82% and specificity of 100%.&amp;#x2028; Discussion: With an imaging penetration depth of 2-3 mm, equivalent to that used for histological assessment, OCT provides unique real-time cellular-level imaging to identify positive and close margins. In these studies, areas of higher scattering tissue with an irregular or heterogeneous pattern were identified, differentiating them from the abundant adipose tissue found in normal breast tissue. The small nucleus to cytoplasm (N/C) ratio is observed with low-scattering adipocytes compared with the larger N/C ratio found from highly-scattering tumor cells. These intraoperative imaging studies have demonstrated the ability for OCT to identify positive surgical margins. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 802.

Effects of novel retinoic acid metabolism blocking agent (VN/14-1) on N-methyl-N-nitrosourea (MNU)-induced mammary carcinoma and uterus in the rat model.

Abstract Abstract #2133 Background: All-trans-retinoic acid (ATRA) and other retinoids play key roles in prevention and therapy of many proliferative diseases including cancers. VN/14-1 [4-(±)-(1H-Imidazol-1-yl)-(E)-retinoic acid], which is a new generation novel retinoic acid metabolism blocking agent (RAMBA), works by inhibiting the breakdown of ATRA. The inhibitory effects of VN/14-1 on the growth of human breast cancer cells and human breast tumors in the nude mouse model have been previously demonstrated. The purpose of this study was to evaluate the effects of VN/14-1 on the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma model, as well as on the uterus in immature ovariectomized (OVX) rats.&amp;#x2028; Methods: (1) VN/14-1 (5, 10, and 20 mg/kg/d) was given by oral gavage to grouped female Sprague Dawley (SD) rats bearing MNU-induced mammary carcinoma for 8 weeks, after which tumor weight and volume, as well as histology were measured. (2) VN/14-1 (10 and 20 mg/kg/d) and b-estradiol (10 mg/kg/d) were given alone or in combination, by oral gavage (VN/14-1) and subcutaneous injection (b-estradiol), to immature OVX SD rats for 3 days, after which uterine weight and histology were measured.&amp;#x2028; Results: (1) At the end of the treatment period, the administration of 5, 10 and 20 mg/kg/d VN/14-1 caused significant reductions of 19.1, 34.4 and 44.3%, respectively, in mean tumor weight compared with the control animals (all p &amp;lt; 0.05). The cumulative tumor growth was also significantly slower in groups receiving 5, 10 and 20 mg/kg/d compared to the control group in a dose-dependent manner. (2) Immature OVX rats given VN/14-1 at doses of 10 and 20 mg/kg, had reduction in uterine wet weight of up to 56% compared to OVX controls (P &amp;lt; 0.001). OVX rats given VN/14-1 of 10 and 20 mg/kg in combination with β-estradiol had reduction in uterine wet weight of up to 58% compared to the OVX rats given β-estradiol alone (P &amp;lt; 0.001). The adverse toxic effects such as fatigue and anorexia were occurred in the groups at high dose of 20 mg/kg.&amp;#x2028; Conclusions: RAMBA VN/14-1 was able to inhibit the growth of tumors in the MNU-induced ER positive rat mammary tumor model and antagonized the stimulatory effect of β-estradiol on the uterus. The studies suggest VN/14-1 might be an effective novel therapy for ER positive breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2133.

Expression order of alpha-v and beta-3 integrin subunits in the N-methyl-N-nitrosourea-induced rat mammary tumor model

Expression order of alpha-v and beta-3 integrin subunits in the N-methyl-N-nitrosourea-induced rat mammary tumor model

Expression Order of Alpha-v and Beta-3 Integrin Subunits in the N-Methyl-N-Nitrosourea-Induced Rat Mammary Tumor Model

We investigated the developmental time course of molecular expression of αvβ3 subunits in a carcinogen-induced rat mammary tumor model for human ductal carcinoma in situ (DCIS). Tumors during various stages of growth (from <0.1 to >2.0 cm) were analyzed immunohistochemically for the expression of the αvβ3 integrin and its subunits. In general, the expression profiles of these integrin subunits were directly proportional to the size of the tumor. The pattern of immunostaining revealed that anti-αvβ3 monoclonal antibody binds to specific sites of tumor sections, forming isolated stained patches. This isolated patch pattern was found in more developed larger tumors. This could be due to the fact that the integrin molecule might be involved in migration and nesting of tumor cells into specific regions to form DCIS or intraductal carcinoma. Results also showed that the αv subunit expresses earlier than the β3 subunit. These data provide insight into tumor cell biology and developmental characteristics that will guide the future construction and use of targeted contrast and therapeutic agents capable of tracking, imaging, or treating a tumor at the earliest stage of formation.

VRP immunotherapy targeting neu: treatment efficacy and evidence for immunoediting in a stringent rat mammary tumor model

The ability to overcome intrinsic tolerance to a strict "self" tumor-associated antigen (TAA) and successfully treat pre-existing tumor is the most stringent test for anti-tumor immunotherapeutic strategies. Although this capacity has been demonstrated in various models using complicated strategies that may not be readily translated into the clinical arena, straightforward antigen-specific immunotherapeutic strategies in the most stringent models of common epithelial cancers have largely failed to meet this standard. We employed an immunotherapeutic strategy using an alphavirus-based, virus-like replicon particle (VRP), which has in vivo tropism for dendritic cells, to elicit immune responses to the non-mutated TAA rat neu in an aggressive rat mammary tumor model. Using this VRP-based immunotherapeutic strategy targeting a single TAA, we generated effective anti-tumor immunity in the setting of pre-existing tumor resulting in the cure of 36% of rats over multiple experiments, P = 0.002. We also observed down-regulation of rat neu expression in tumors that showed initial responses followed by tumor escape with resumption of rapid tumor growth. These responses were accompanied by significant anti-tumor proliferative responses and CD8+ cellular tumor infiltrates, all of which were restricted to animals receiving the anti-neu immunotherapy. Together these data, obtained in a stringent "self" TAA model, indicate that the VRP-based antigen-specific immunotherapy elicits sufficiently potent immune responses to exert immunologic pressure, selection, and editing of the growing tumors, thus supporting the activity of this straightforward immunotherapy and suggesting that it is a promising platform upon which to build even more potent strategies.

A novel steroidal selective steroid sulfatase inhibitor KW-2581 inhibits sulfated-estrogen dependent growth of breast cancer cells in vitro and in animal models

We screened a series of 17beta-(N-alkylcarbamoyl)-estra-1,3,5(10)trine-3-O-sulfamate derivatives, and describe here a potent and selective steroid sulfatase (STS) inhibitor with antitumor effects in breast cancer models in vitro and in vivo. In biochemical assays using crude enzymes isolated from recombinant Chinese hamster ovary cells expressing human arylsulfatses (ARSs), one of the best compounds, KW-2581, inhibited STS activity with an IC(50) of 4.0 nM, while > 1000-fold higher concentrations were required to inhibit the other ARSs. The failure to stimulate the growth of MCF-7 human breast cancer cells as well as in uteri in ovariectomized rats indicated the lack of estrogenicity of this compound. In MCF-7 cells transfected with the STS gene, termed MCS-2 cells, KW-2581 inhibited the growth of cells stimulated by estrone sulfate (E1S) but also 5-androstene-3beta, 17beta-diol 3-sulfate (ADIOLS) and dehydroepiandrostenedione 3-sulfate. We found that oral administration of KW-2581 inhibited both E1S- and ADIOLS-stimulated growth of MCS-2 cells in a mouse hollow fiber model. In a nitrosomethylurea-induced rat mammary tumor model, KW-2581 induced regression of E1S-stimulated tumor growth as effectively as tamoxifen or another STS inhibitor, 667 Coumate. Dose-response studies in the same rat model demonstrated that more than 90% inhibition of STS activity in tumors was necessary to induce tumor shrinkage. STS activity in tumors has well correlated with that in leukocytes, suggesting that STS activity in leukocytes could be used as an easily detectable pharmacodynamic marker. These findings demonstrate that KW-2581 is a candidate for development as a therapeutic agent for the treatment of hormone receptors-positive breast cancer.

Synthesis and preliminary chemotherapeutic evaluation of the fully C-linked glucuronide of N-(4-hydroxyphenyl)retinamide

All- trans retinoic acid analogues such as N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventive and chemotherapeutic agents but their utility has been hampered by dose-limiting side effects. The glucuronide derivatives of 4-HPR, the oxygen-linked 4-HPROG and the carbon-linked 4-HPRCG, have been found to be more effective agents. The synthetic route to the fully C-linked analogue of 4-HPROG (4-HBRCG), which employs Suzuki coupling and Umpolung chemistries as key methodologies, is shown. The results of this study show 4-HBRCG to be an effective chemotherapeutic agent in a rat mammary tumor model while being devoid of classical retinoid toxicities.