DMBA-induced Tumors Research Articles

Abstract C135: AZ’4425 is a potent, selective, and orally bio-available progesterone receptor antagonist that has shown anti-tumor activity and inhibition of cancer stem cell proliferation.

Abstract Progesterone is an important hormone in breast cancer; the paracrine mediator of the progesterone receptor, RANKL plays a critical role in cancer stem cells, metastasis and tumorigenesis. Steroidal inhibitors of PR have shown modest clinical activity but have been limited by toxicity. AZ’4425 is a novel, potent non-steroidal progesterone receptor (PR) antagonist which selectively inhibits PR compared to other steroid hormone receptors (∼ 1000-fold ER, MR, GR, AR). AZ’4425 competitively inhibits progesterone binding, thus preventing progesterone-induced phosphorylation of PR, nuclear translocation and PR-induced transcription with an IC50 of 26nM. Anchorage independent growth of T47D cells is similarly inhibited with an IC50 of 25nM. Using cancer stem cell (CSC) assays (mammosphere formation and ALDH positivity) we have shown broad cellular activity in both ER/PR positive tumor cell lines and early and late stage clinical samples. Furthermore, AZ’4425 prevents the increase in CSCs, and miRNA changes (miR221/2 and miR200c) induced by anti-estrogen therapies, suggesting a potential role in prevention of acquired resistance. In vivo, the PR antagonist inhibits transcription of progesterone-induced genes including RANKL and SGK1. Notably, AZ’4425 results in significant inhibition of new tumor formation and results in stasis and regression of DMBA-induced tumors, both as monotherapy and in combination with letrozole. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C135. Citation Format: Christine M. Chresta, Denis Alferez, Georgia Cerillo, Emma Still, Adina Hughes, Graeme Walker, Jane Kendrew, Claire Sadler, Jayne Harris, Iain Simpson, Andrew P. Thomas, Al Rabow, Robert Clarke, Sacha Howell, Graham Richmond. AZ’4425 is a potent, selective, and orally bio-available progesterone receptor antagonist that has shown anti-tumor activity and inhibition of cancer stem cell proliferation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C135.

Characterization of synergistic anti-cancer effects of docosahexaenoic acid and curcumin on DMBA-induced mammary tumorigenesis in mice

BackgroundThe major obstacles to the successful use of individual nutritional compounds as preventive or therapeutic agents are their efficacy and bioavailability. One approach to overcoming this problem is to use combinations of nutrients to induce synergistic effects. The objective of this research was to investigate the synergistic effects of two dietary components: docosahexaenoic acid (DHA), an omega-3 fatty acid present in cold-water fish, and curcumin (CCM), an herbal nutrient present in turmeric, in an in vivo model of DMBA-induced mammary tumorigenesis in mice.MethodsWe used the carcinogen DMBA to induce breast tumors in SENCAR mice on control, CCM, DHA, or DHA + CCM diets. Appearance and tumor progression were monitored daily. The tumors were harvested 15 days following their first appearance for morphological and immunohistological analysis. Western analysis was performed to determine expression of maspin and survivin in the tumor tissues. Characterization of tumor growth was analyzed using appropriate statistical methods. Otherwise all other results are reported as mean ± SD and analyzed with one-way ANOVA and Tukey’s post hoc procedure.ResultsAnalysis of gene microarray data indicates that combined treatment with DHA + CCM altered the profile of “PAM50” genes in the SK-BR-3 cell line from an ER-/Her-2+ to that resembling a “normal-like” phenotype. The in vivo studies demonstrated that DHA + CCM treatment reduced the incidence of breast tumors, delayed tumor initiation, and reduced progression of tumor growth. Dietary treatment had no effect on breast size development, but tumors from mice on a control diet (untreated) were less differentiated than tumors from mice fed CCM or DHA + CCM diets. The synergistic effects also led to increased expression of the pro-apoptotic protein, maspin, but reduced expression of the anti-apoptotic protein, survivin.ConclusionsThe SK-BR-3 cells and DMBA-induced tumors, both with an ER- and Her-2+ phenotype, were affected by the synergistic interaction of DHA and CCM. This suggests that the specific breast cancer phenotype is an important factor for predicting efficacy of these nutraceuticals. The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon the molecular phenotype of the cancer.

Screening of chemopreventive effect of naringenin-loaded nanoparticles in DMBA-induced hamster buccal pouch carcinogenesis by FT-IR spectroscopy

The aim of the present study is to investigate the chemopreventive effects of the prepared naringenin-loaded nanoparticles (NARNPs) relative to efficacy of free naringenin (NAR) in modifying the functional, structural, and compositional changes at the molecular level during 7, 12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis by Fourier transform infrared (FT-IR) spectroscopy. The results revealed that a significant increase in the amount of proteins and nucleic acid contents and a decrease in the amount of lipids and glycogen contents are observed in DMBA-induced tumor tissues. In addition, in tumor tissues a decrease in lipid order and a significant increase in membrane dynamics were noticed. Further, the composition and secondary structure of proteins were found to be altered, which indicates some important structural alterations in the existing proteins and/or the expression of new types of proteins occurring under the tumor transformation. Furthermore, oral administration of free NAR and NARNPs significantly increased lipids and their order as well as increased the glycogen contents and decreased the levels of proteins and nucleic acid contents. On a comparative basis, NARNPs were found to have a more potent antitumor effect than free NAR in completely preventing the formation of squamous cell carcinoma and in improving the biochemical constituents to a normal range in DMBA-induced HBP carcinogenesis. The present study further shows a great potential of FT-IR spectroscopy as a complimentary tool for the screening of various anticancer drugs and follow-up, which may allow faster response to critical problems arising during treatment.

Modulating Effect of Lupeol on the Expression Pattern of Apoptotic Markers in 7, 12-Dimethylbenz(a)anthracene Induced Oral Carcinogenesis

Apoptosis, also known as cell suicide or programmed cell death, removes unwanted and genetically damaged cells from the body. Evasion of apoptosis is one of the major characteristic features of rapidly proliferating tumor cells. Chemopreventive agents inhibit or suppress tumor formation through apoptotic induction in target tissues. The aim of the present study was to investigate the pro-apoptotic potential of lupeol during 7,12-dimethylbenz(a) anthracene (DMBA) induced hamster buccal pouch carcinogenesis. Topical application of 0.5% DMBA three times a week for 14 weeks in the buccal pouches of golden Syrian hamsters resulted in oral squamous cell carcinoma. The expression pattern of apoptotic markers was analyzed using immunohistochemistry (p53, Bcl-2, Bax) and ELISA reader (caspase 3 and 9). In the present study, 100% tumor formation with defects in apoptotic markerexpression pattern was noticed in hamsters treated with DMBA alone. Oral administration of lupeol at a dose of 50 mg/kg bw completely prevented the formation oral tumors as well as decreased the expression p53 and Bcl-2, while increasing the expression of Bax and the activities of caspase 3 and 9. The present study thus indicated that lupeol might inhibit DMBA-induced oral tumor formation through its pro-apoptotic potential in golden Syrian hamsters.

Oral Delivery of Doxorubicin Using Novel Polyelectrolyte-Stabilized Liposomes (Layersomes)

The present study explores the potential of polyelectrolyte-coated liposomes for improving the oral deliverability of doxorubicin (Dox). As a part of formulation strategy, stearyl amine was selected as a formulation component to provide positive charge to liposomes, which were subsequently coated with anionic poly(acrylic acid) (PAA) followed by coating of cationic polyallyl amine hydrochloride (PAH) in a layer by layer manner and led to the formation of a robust structure "layersomes". Optimization of various process variables were carried out, and optimized formulation was found to have particle size of 520.4 ± 15.0 nm, PDI of 0.312 ± 0.062, ζ potential of +30.4 ± 5.32 mV, and encapsulation efficiency of 63.4 ± 4.26%. Layersomes were not only stable in simulated gastrointestinal fluids but also presented sustained drug release (∼35%) as compared to both Dox-liposomes and PAA-Dox-liposomes (∼67%), the release pattern being Higuchi kinetics. The in vivo pharmacokinetics studies revealed about 5.94-fold increase in oral bioavailability of Dox as compared to free drug. In vivo antitumor efficacy in a DMBA-induced breast tumor model further exhibited significant reduction in the tumor growth as compared to control and IV-Dox, while results were comparable to IV-LipoDox. Layersomes also exhibited a marked reduction in cardiotoxicity in comparison with IV-doxorubicin and IV-LipoDox (marketed formulation), as evidenced by the reduced levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), and creatine phosphokinase (CK-MB) and increased levels of glutathione (GSH) and superoxide dismutase (SOD). The reduced cardiotoxicity of layersomes was further confirmed by comparative histopathological examination of heart tissue after treatment with various formulations. The positive results of the study strengthen our expectation that the developed formulation strategy can be fruitfully exploited to improve the oral deliverability of poorly bioavailable drugs and can open new vistas for oral chemotherapy.

Therapeutic effect of ovarian intra-arterial infusion of GE7-delivery system-mediated HSVl-tk/ganciclovir gene therapy in a rat model of malignant ovarian tumor

To observe the gene expression of herpes simplex virus type 1 thymidine kinase (HSVl-tk) in rat malignant ovarian tumor tissues and the therapeutic effect of ganciclovior (GCV) after intra-arterial infusion of HSVl-tk gene therapy mediated by GE7-delivery system. A GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 4-element complex was constructed. Eighteen rats with DMBA-induced ovarian tumor were divided into 3 groups as Atk, ANS and Vtk groups. The 4-element complex GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 was injected via the ovarian artery into the rats of Atk group, saline buffer was injected in the ANS groups, and the 4-element complex was injected via the tail vein into the rats of Vtk group. All rats received intraperitoneal injection of GCV in a dose of 50 mg/kg daily for 10 days. The rats were sacrificed 3 days after the final dose of GCV, and the tumor weight was measured and tumor growth inhibition rate was calculated. Flow cytometry was used to assess the cell cycle and apoptosis. The tumor weight in the rats of Atk group was (4.77 ± 2.31) g, significantly lower than that of ANS group [(14.66 ± 6.26) g, P < 0.01] and Vtk group [(17.53 ± 7.19) g, P < 0.01]. The tumor growth inhibition rate of the Atk group was 67.5%, while that of Vtk group was -19.6%. The flow cytometry showed that S-phase tumor cells in the Atk group were (54.32 ± 9.65)%, significantly higher than that in the ANS (27.43 ± 9.22)% and (30.16 ± 11.57)% in the Vtk group (both P < 0.01). The tumor cell apoptosis rate in the Atk group was (39.15 ± 12.16)%, significantly higher than that in the ANS group [(11.86 ± 5.28)%, P < 0.01] and Vtk group [(14.32 ± 6.43)%, P < 0.01]. HSV1-tk/GCV gene therapy system mediated by GE7 non-viral delivery system via ovarian arterial infusion effectively causes cell cycle arrest at S phase and enhances cell apoptosis, therefore, exerts an inhibitory effect on tumor growth.

Therapeutic and Preventive Effects of an Epidermal Growth Factor Receptor Inhibitor on Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is the sixth most common human neoplasm worldwide. Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is highly expressed in human OSCC and is a target for cancer therapy and prevention. This study investigated the therapeutic and preventive effects of an inhibitor of EGFR (PD153035) on OSCC. The effects of PD153035 were examined in human cancer cell lines in vitro, in an athymic nude mouse xenograft model in vivo, and in the 7,12-dimethyl benz[a]anthracene (DMBA)-induced hamster cheek pouch tumour model in vivo. PD153035 significantly inhibited cell growth, delayed cell cycle progression and induced apoptosis in human OSCC cells in vitro. In vivo, PD153035 inhibited xenograft tumour growth in nude mice in a dose-dependent manner and prevented the development of OSCC at the postinitiation stage in the DMBA-induced hamster cheek pouch tumour model. PD153035 inhibited the DMBA-induced increases in cell proliferation and in levels of phosphorylated EGFR and phosphorylated signal transducer and activator of transcription 3 (STAT3) protein in the hamster cheek pouch. Inhibitors of EGFR, such as PD153035, have potential value in the treatment and prevention of OSCC.

Estetrol prevents and suppresses mammary tumors induced by DMBA in a rat model

Estetrol (E4) is a pregnancy-specific estrogenic steroid hormone produced by the human fetal liver in both male and female fetuses. During pregnancy, E4 plasma values increase exponentially until parturition and decrease thereafter. The synthesis of E4 in the liver of a newborn ceases during the first weeks after birth. Here we report the effect of E4 on the initiation and growth of mammary tumors chemically induced by 7,12 dimethylbenz(a)anthracene (DMBA) in female Sprague-Dawley rats in two different protocols. Two prevention studies to test the effect on initiation and growth of induced tumors and one intervention study to test the effect on tumor growth were performed. In the prevention studies, the effect of oral doses of E4 over a dose range of 0.5-3.0 mg/kg was investigated. In the intervention study, oral doses of 1, 3 and 10 mg/kg E4 were used. The anti-estrogen tamoxifen (TAM) and ethinylestradiol (EE) were used as reference compounds. In all studies, a group with ovariectomized animals (OVX) was included. In the prevention studies, 2.5 mg and 3 mg/kg E4 showed a significant effect on the number and growth of induced tumors by DMBA, and the effects were comparable to those of TAM, whereas EE had no effect. In the intervention study, the effect of a high dose of E4 (10 mg/kg) on tumor number was similar to that of OVX and better than TAM and high-dose EE. The 3 mg/kg E4 had an effect comparable to high-dose EE. The treatment effects were largely due to complete regression of existing tumors. The natural fetal estrogen E4 prevents tumor initiation by DMBA and inhibits the growth of existing DMBA-induced tumors.

Inhibitory Effects of Combination of Lycopene and Genistein on 7,12- Dimethyl Benz(a)anthracene-Induced Breast Cancer in Rats

Breast cancer is one of the most common cancers in women. Carotenoids and soy isoflavones have been postulated to have breast cancer preventive effects. We investigated the potential preventive effects of lycopene and genistein, alone and in combination, on breast cancer development in female Wistar rats treated with 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogen known to induce breast tumors. Mammary carcinogenesis was initiated by a single, oral gavage of DMBA (80 mg/kg body weight) at 55 days of animal age. Fifty female Wistar rats were divided into 5 experimental groups having 10 animals per group: Group 1 (normal control), Group 2 (DMBA control), Group 3 (DMBA + lycopene), Group 4 (DMBA + genistein), and Group 5 (DMBA + lycopene and genistein). Rats were fed either lycopene (20 mg /kg bw) or genistein (2 mg /kg bw) by oral gavage (3 times per week) starting 2 wk prior to DMBA injection. Treatment was continued for 20 wk. Rats treated with DMBA developed mammary tumors with 100% tumor incidence during the 20-wk study. Inhibition of mammary cancer incidence by lycopene (70%), genistein (60%) and their combination (40%) was observed. Tumor weight decreased by 48%, 61%, and 67%, and mean tumor volume decreased by 18%, 35%, and 65% with lycopene, genistein, and lycopene + genistein, respectively (P < 0.01 for the combination). The proportions of adenocarcinoma masses decreased with lycopene and genistein combination (P < 0.05). Administration of lycopene and genistein combination suppressed breast cancer development and was associated with a decrease in MDA, 8-isoprostane, and 8-OhdG levels and with an increase in serum lycopene and genistein levels. Animals administered DMBA developed breast cancer, which was associated with increased expression of Bcl-2 and decreased expression of Bax, caspase 3, and caspase 9 in mammary tissues. Administration of genistein and lycopene in combination was more effective in inhibiting DMBA-induced breast tumors and modulating the expression of apoptosis associated proteins than the administration of each agent alone. Our results suggest that lycopene and genistein are potent antioxidants and, when given in combination, offer maximum protection against DMBA-induced mammary carcinogenesis.

Abstract C31: DMBA-induced skin tumors in HPV16 E7 transgenic mice are affected by bovine α-lactalbumin made lethal to tumor cells (BAMLET)

Abstract Background and Purpose: Human papillomavirus Type 16 (HPV16) infection is necessary but not alone a sufficient causal risk factor for cervical and other anogenital cancers and nearly 25% of head and neck malignancies (1, 2). HPV16 E7 is one of the important oncoproteins that promotes carcinogenesis. Most treatments for cervical precancers are ablative and few therapeutic drugs are available. Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET), a complex of Human α-Lactalbumin and oleic acid shows promise for treating skin papillomas by differentially inducing apoptosis in transformed cells without adverse effects on normal cells. Bovine α-Lactalbumin Made Lethal to Tumor Cells (BAMLET) has been shown to be biologically equivalent to HAMLET (3) and in vitro studies suggest BAMLET and HAMLET show similar cytotoxic effects in tumor cell lines (4). Further, in vitro studies suggest both a dose- and time-dependent response to BAMLET treatment (3–6). To determine the effect of BAMLET on tumor number, size and histological characteristics in vivo, we compared two groups of BAMLET-treated with normal saline (NS)-treated HPV16 E7 transgenic C57/BL6 mice (TG) and syngeneic mice receiving chemical carcinogen DMBA topically to induce skin tumor. Methods: For 56 TG and syngeneic mice, in six treatment groups, 100nmol/200μl DMBA was applied topically on the dorsal thoracic flank for up to 20 weeks. BAMLET, 0.7 mM, was applied daily for 7d before and either 11d (18d BAMLET) or up to 23 weeks (24w BAMLET) following DMBA initiation. As a comparison, NS was similarly applied to 10 TG and syngeneic mice for a total of 24 weeks. A series of bivariate zero-inflated repeated-measures Poisson (ZIP) models predicted the number of papillomas based on TG/syngeneic groupings, and treatment group. The fully adjusted model evaluated the effect of the TG, treatment group and duration, and time on study for predicting the number of tumors. Least-squares linear regression (LSLR) was used to estimate the effect of treatment on average tumor size at censoring or death, controlling for the effect of gender, among the transgenic mice. Results: On average, papillomas developed after 9 weeks of DMBA treatment. Analyses showed TG-mice developed more tumors than did syngeneic mice (p&amp;lt;0.0001), controlling for the effect of time on study (p&amp;lt;0.0001). In the fully adjusted analyses, the number of tumors was most affected by the TG (p&amp;lt;0.0001) and time on study (p&amp;lt;0.0001), but treatment showed no effect: 18d (p=0.9) and 24w (p=0.8) vs. normal saline (NS). Among transgenic mice, time on study was shorter than for syngeneic comparators and was a proxy indicator across treatment groups, confounding the ZIP analyses. Among transgenic mice, BAMLET treatment predicted average tumor size at censoring or death in the LSLR analysis. Specifically, among transgenic mice, the average tumor size was greatest for the 18d (μ=0.24 cm, p=0.003) and 24w BAMLET-treated (μ=0.20 cm, p=0.03) mice when compared to NS-treated controls (μ=0.12 cm). Discussion and Conclusion: These preliminary data suggest the effect of the transgene is strong on the number of tumors the result from DMBA treatment and on survival. This strong effect suggests we lack the power to detect an effect of BAMLET in transgenic mice. However, some data herein suggest BAMLET may increase average tumor size among HPV16-E7 transgenic mice. In light of positive findings in human clinical studies using the human analogue, HAMLET, understanding whether the effects of α-Lactalbumins and oleic acid complexes are modulated by HPV E6, E7, or E6/E7 oncogenes warrants further exploration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C31.

Effect of liposomal celecoxib on proliferation of colon cancer cell and inhibition of DMBA-induced tumor in rat model.

Celecoxib, a selective cyclooxygenase-2 inhibitor, has shown potential anticancerous activity against majority of solid tumors especially on patients with colon cancer. However, associations of serious side effects limit the usage of celecoxib in colon cancer treatment. To address this issue and provide an alternative strategy to increase the efficacy of celecoxib, liposomal formulation of celecoxib was prepared and characterized. Anticancer activity of liposomal celecoxib on colon cancer cell HCT 15 was evaluated in vitro. Furthermore, tumor inhibition efficiency by liposomal celecoxib was studied on 7,12-dimethyl benz(a)anthracene (DMBA)-induced tumor in rat model. In order to elucidate the antioxidant activity of celecoxib-loaded liposomes, antioxidant superoxide dismutase (SOD) generation and lipid peroxide (LPx) formation in both liver and kidney tissues were examined. Characterization of the formed unilamellar liposomes revealed the formation of homogeneous suspension of neutral (empty) or anionic (celecoxib-loaded) liposomes with a well-defined spherical shape which have a mean size of 103.5 nm (empty liposome) and 169 nm (liposomal celecoxib). High-performance liquid chromatography (HPLC) analysis and hemolytic assay demonstrated 46% of celecoxib entrapment efficiency and significantly low hemolysis, respectively. Liposomal celecoxib exhibited dose-dependent cytotoxicity and apoptotic activity against HCT 15 cells which are comparable to free celecoxib. In vivo study demonstrated inhibition of tumor growth. Biochemical analysis of the liposomal celecoxib-treated group significantly inhibited the LPx formation (oxygen-free radicals) and increased the activity of SOD. Our results present the potential of inhibiting colon cancer in vitro and DMBA-induced tumor in rat model in vivo by liposomal celecoxib.

Bisphenol A Increases Mammary Cancer Risk in Two Distinct Mouse Models of Breast Cancer

Bisphenol A (BPA) is an industrial plasticizer that leaches from food containers during normal usage, leading to human exposure. Early and chronic exposure to endocrine-disrupting environmental contaminants such as BPA elevates the potential for long-term health consequences. We examined the impact of BPA exposure on fetal programming of mammary tumor susceptibility as well as its growth promoting effects on transformed breast cancer cells in vivo. Fetal mice were exposed to 0, 25, or 250 μg/kg BPA by oral gavage of pregnant dams. Offspring were subsequently treated with the known mammary carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). While no significant differences in postnatal mammary development were observed, both low- and high-dose BPA cohorts had a statistically significant increase in susceptibility to DMBA-induced tumors compared to vehicle-treated controls. To determine if BPA also promotes established tumor growth, MCF-7 human breast cancer cells were subcutaneously injected into flanks of ovariectomized NCR nu/nu female mice treated with BPA, 17beta-estradiol, or placebo alone or combined with tamoxifen. Both estradiol- and BPA-treated cohorts formed tumors by 7 wk post-transplantation, while no tumors were detected in the placebo cohort. Tamoxifen reversed the effects of estradiol and BPA. We conclude that BPA may increase mammary tumorigenesis through at least two mechanisms: molecular alteration of fetal glands without associated morphological changes and direct promotion of estrogen-dependent tumor cell growth. Both results indicate that exposure to BPA during various biological states increases the risk of developing mammary cancer in mice.

Abstract 1844: Apigenin prevents development of progestin-accelerated 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors in Sprague-Dawley rats

Abstract Post-menopausal women undergoing hormone replacement therapy (HRT) containing both progestins (P) and estrogens (E) are at an increased risk of developing breast cancer compared with those taking E alone. The biological mechanisms behind this phenomenon however, remain obscure. We showed that medroxyprogesterone acetate (MPA), a commonly used P, increases the production of the potent angiogenic growth factor VEGF in breast cancer cells that was blocked by the anti-progestin RU-486 suggesting the involvement of progesterone receptors (Int J Cancer, 2001, 92:469). This led to our hypothesis that P increases the angiogenic potential of latent tumorigenic cells in the breast, leading to development of palpable tumors. To prove this hypothesis, we developed in vivo models with human xenografts in mice (Cancer Res., 2007, 67:9929), and DMBA-induced tumors in rats (Clin Can Res, 2006, 12:4062). In both models, MPA accelerated tumor development that was blocked by RU-486. Since RU-486 has severe side-effects, we undertook studies aimed at identifying less toxic naturally-occurring compounds that might prevent P-dependent VEGF induction and tumor acceleration. We recently identified apigenin, a low molecular weight flavonoid commonly found in fruits and vegetables, which inhibited MPA-induced VEGF secretion from human breast cancer cells (Menopause, 2010, 17:1055). In this study we used the DMBA-induced mammary tumor model to determine whether apigenin prevents the development of P-accelerated tumors in vivo. Female Sprague-Dawley rats were treated with DMBA by oral gavage (Day 1). Seven days prior to implantation of 25 mg/60 day release MPA pellets (Day 28), ip administration of apigenin (50 mg/kg daily) began, and continued for 10 days. Animals were palpated for tumors every second day and experiment was terminated 60 days after DMBA treatment was initiated. Apigenin treatment led to a significant delay in the appearance of the first tumor, compared with MPA alone (56 vs 41 days; p&amp;lt;0.05, LIFETEST), as well as a reduction in tumor incidence and tumor multiplicity. Immunohistochemcial analysis with mammary tissue collected immediately after apigenin administration ceased revealed that even though apigenin decreased the overall incidence of MPA-accelerated tumors, it did not block or reverse DMBA-induced mammary tissue hyperplasia, suggesting that the effects of the flavonoid are exerted at a specific stage of tumor development. Further studies showed that apigenin suppressed both VEGF and its receptor, VEGFR-2, in the hyperplastic regions of the mammary gland. Thus apigenin appears to be a compound with useful anti-tumor and anti-angiogenic properties which could be given to post-menopausal women to prevent development of P-accelerated tumors. Support: COR award from the College of Vet Medicine and by research funds from RADIL, Univ. of Missouri. 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 1844. doi:10.1158/1538-7445.AM2011-1844

Chemopreventive Effect of Nonsteroidal Anti-inflammatory Drugs on 9,10-Dimethylbenz[a]anthracene-Induced Lung Carcinogenesis in Mice

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, celecoxib, and etoricoxib were studied as chemopreventive agents in lung cancer in mice induced by 9,10-dimethylbenz[a]anthracene (DMBA). The animals were subjected to a single intratracheal instillation of DMBA by surgical intervention, while they were treated with oral NSAIDs daily at their following anti-inflammatory dose: aspirin 25 mg/kg, celicoxib 6 mg/kg, and etoricoxib 0.6 mg/kg body weight, respectively. The animals were sacrificed after 18 weeks of treatment. Results showed a significant incidence of pulmonary tumors, dysplastic changes in histopathology, and signs of inflammatory occurrence in the DMBA-treated animals, which were grossly reversed by the NSAIDs. A greater number of macrophages, neutrophils, and lymphocytes were seen in the bronchoalveolar lavage (BAL) smear while the inflammatory cell counts decreased in DMBA + NSAIDs groups. A significant increase in the drug-metabolizing enzymes viz. cytochrome p450, cytochrome b5, and glutathione-S-transferase was noted in the DMBA group, which was reverted back in the NSAID-treated mice. Similarly, the subcelluler enzymes were elevated in DMBA, but significantly fell in the NSAID groups. DMBA also caused a higher level of lipid peroxidation as well as the different antioxidant enzyme activity, which were corrected by the NSAIDs. A marked elevation was noticed in the total lipid composition and its individual constituents in the DMBA group, which was reverted back appreciably by the NSAIDs. The results suggest that the DMBA-induced lung tumor development in balb/c mice could be a reliable model to test the chemopreventive potential of the NSAIDs.

Effects of Rich-in-Fat Diets and Highly Selective COX-2 Inhibitors on 7,12-Dimethylbenz-(A)-Anthracene-Induced Tumor Growth

The effects of diet, of non-steroidal anti-inflammatory drugs, or of their combination on carcinogenesis continue to be a case for controversy. Diets that are high in fat have been linked to increased risk of various tumors. At the same time there is substantial, but not conclusive, evidence that the risk of breast and colon cancer correlates with total fat intake rather than a specific type of fat. On the other hand, non-steroidal anti-inflammatory drugs (NSAIDs) have been studied extensively because they appear to delay or inhibit the development of malignant and pre-malignant lesions. 7,12-Dimethylbenz-(a)-anthracene (DMBA) has been used for a long time to induce carcinogenesis in a number of rat animal models. The present study attempts to identify the effects on DMBA-induced tumor growth (a) of diets rich in fat and (b) of the highly selective COX-2 inhibitor Celecoxib, which has been claimed to offer substantial protection against carcinogenesis.

Regression of progestin-accelerated 7,12-dimethylbenz[a]anthracene-induced mammary tumors in Sprague–Dawley rats by p53 reactivation and induction of massive apoptosis: a pilot study

p53 Reactivation and induction of massive apoptosis (PRIMA-1) is a small-molecule compound that reactivates mutant p53, restoring its normal tumor suppressor function. PRIMA-1 effectively suppresses the growth of homogeneous p53-deficient tumor xenografts in immunosuppressed mice; however, the ability of PRIMA-1 to suppress the growth of mammary tumors in rodents and other species is not well characterized. Here, we examined the ability of PRIMA-1 to suppress the growth of 7,12-dimethylbenz[a]anthracene (DMBA)-induced and progestin-accelerated DMBA-induced mammary tumors in Sprague-Dawley rats. Mammary tumors were induced in female rats with DMBA or DMBA plus progestin treatment. After tumors had reached 5-25 mm(2) in size, PRIMA-1 was administered twice a day for 3 days via tail vein injection (20 or 50 mg/kg). Tumor size was monitored every day following PRIMA-1 for at least 15 days prior to killing. PRIMA-1 caused regression of approximately 40% of progestin-accelerated DMBA-induced mammary tumors, but did not induce regression of native non-progestin-accelerated DMBA-induced tumors. Importantly, PRIMA-1 also suppressed the emergence of any new progestin-accelerated tumors in this model. Immunological studies with an antibody that selectively reacts with mutant p53 suggested that none of the native DMBA-induced tumors expressed mutant p53. By contrast, six out of eight progestin-accelerated DMBA-induced tumors stained for mutant p53 protein. In PRIMA-1-treated tumor-bearing rats, tumor regression correlated with conversion of mutant to wild-type p53 conformation, reduced expression of vascular endothelial growth factor and estrogen receptor, lack of blood vessel perfusion, increased expression of p21, and massively increased expression of anti-angiogenic protein, secreted protein acidic and rich in cysteine. These pre-clinical results suggest that PRIMA-1, as a single agent or in combination with other anti-cancer compounds, has potential as a novel chemotherapeutic treatment for progestin-accelerated human breast cancer.

Differential Gene Signatures in Rat Mammary Tumors Induced by DMBA and Those Induced by Fractionated γ Radiation

The aim of this work was to identify specific genes involved in rat mammary tumors induced by dimethylbenz(a)anthracene (DMBA) or radiation. More TUNEL- and PCNA-positive cells were present in mammary tumors induced by radiation than in tumors induced by DMBA, whereas DNA damage responses like p53 accumulation and histone H2AX phosphorylation were higher in DMBA-induced tumors, even though the pathology was similar in both types of tumors. cDNA microarray and real-time RT-PCR analysis of radiation- or DMBA-induced tumor tissues, revealed that stanniocalcin 2 (Stc2), interferon regulatory factor 1 (Irf1), interleukin 18 binding protein (Il18bp), and chloride channel calcium activated 3 (Clca3) were expressed in both, and that arachidonate 5-lipoxygenase activating protein 1 (Alox5ap) and cathepsin S (Ctss) were expressed only in radiation-induced tumors. No DMBA-specific gene signatures were found. Soft agar growth assays were carried out to identify the carcinogenic features of these specific genes. Cells stably transfected with Alox5ap, Ctss, Stc2, Irf1, Il18bp and Clca3 showed morphological changes compared to controls. These findings indicate different gene alterations in carcinogen- or radiation-induced mammary tumors with similar pathological stages.

Absence of Ras mutations in rat DMBA‐induced mammary tumors

Animal cancer models reduce genetic background heterogeneity and thus, may facilitate identification and analysis of specific genetic aberrations in tumor cells. Rat and human mammary glands have high similarity in physiology and show comparable hormone responsiveness. Thus, spontaneous and carcinogen (e.g., NMU and DMBA)-induced rat mammary models are valuable tools for genetic studies of breast cancer. In NMU-induced rat mammary tumors, activating mutations in Hras codon 12 have frequently been reported and are supposed to contribute to the mammary carcinogenic process. Involvement of Ras mutations in DMBA-induced tumors is less clear. In the present study we investigated the mutation status of the three Ras genes, Hras, Kras, and Nras, in DMBA-induced rat mammary tumors. We examined codons 12, 13, and 61 of all three genes for mutations in 71 tumors using direct sequencing method that in experimental conditions is sensitive enough to detect single nucleotide mutations even when present in only 25% of the test sample. No activating Ras gene mutation was found. Thus, in contrast to NMU-induced rat mammary tumor, tumorigenesis in DMBA-induced rat mammary tumors seems to be independent on activating mutations in the Ras genes. Our finding suggests that the genetic pathways selected in mammary tumor development are influenced by and perhaps dependent on the identity of the inducing agent, again emphasizing the importance of tumor etiology on the genetic changes in the tumor cells.

Dietary turmeric modulates DMBA-induced p21 ras, MAP kinases and AP-1/NF-κB pathway to alter cellular responses during hamster buccal pouch carcinogenesis

The chemopreventive efficacy of turmeric has been established in experimental systems. However, its mechanism(s) of action are not fully elucidated in vivo. The present study investigates the mechanism of turmeric-mediated chemoprevention in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis at 2, 4, 6, 10 and 12 weeks. Dietary turmeric (1%) led to decrease in DMBA-induced tumor burden and multiplicity, and enhanced the latency period in parallel, to its modulatory effects on oncogene products and various cellular responses during HBP tumorigenesis. DMBA-induced expression of ras oncogene product, p21 and downstream target, the mitogen-activated protein kinases were significantly decreased by turmeric during HBP carcinogenesis. Turmeric also diminished the DMBA-induced mRNA expression of proto-oncogenes (c- jun, c- fos) and NF-κB, leading to decreased protein levels and in further attenuation of DMBA-induced AP-1/NF-κB DNA-binding in the buccal pouch nuclear extracts. Besides, buccal pouch of hamsters receiving turmeric diet showed significant alterations in DMBA-induced effects: (a) decrease in cell proliferation (diminished PCNA and Bcl2 expression), (b) enhanced apoptosis (increased expression of Bax, caspase-3 and apoptotic index), (c) decrease in inflammation (levels of Cox-2, the downstream target of AP-1/NF-κB, and PGE2) and (d) aberrant expression of differentiation markers, the cytokeratins (1, 5, 8, and 18). Together, the protective effects of dietary turmeric converge on augmenting apoptosis of the initiated cells and decreasing cell proliferation in DMBA-treated animals, which in turn, is reflected in decreased tumor burden, multiplicity and enhanced latency period. Some of these biomarkers are likely to be helpful in monitoring clinical trials and evaluating drug effect measurements.

In Vitro Antioxidative Potential of Lactoferrin and Black Tea Polyphenols and Protective Effects In Vivo on Carcinogen Activation, DNA Damage, Proliferation, Invasion, and Angiogenesis During Experimental Oral Carcinogenesis

The present study was designed to evaluate the in vitro antioxidant potential of bovine lactoferrin (bLF) and black tea polyphenols [Polyphenon-B (P-B)] as well as in vivo inhibitory effects on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinomas. Antioxidant activity was screened using a panel of assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), hydroxyl radical anion (OH*), superoxide anion (O2*-), and nitric oxide (NO) radical scavenging assays as well as assay for reducing power. The chemopreventive potential of bLF and P-B was assessed in the HBP model based on the modulatory effects on DMBA-induced oxidative DNA damage as well as the expression of proteins associated with carcinogen activation (CYP1A1, CYP1B1), cell proliferation [cyclin D1, proliferating cell nuclear antigen (PCNA), glutathione S-transferase pi (GST-P)], angiogenesis [vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1)], and invasion and metastasis [matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of MMP-2 (TIMP-2)]. Both bLF and P-B showed high radical scavenging activity and reductive potential. Although administration of bLF and P-B alone suppressed DMBA-induced HBP tumors, combined administration of bLF and P-B was more effective in inhibiting HBP carcinogenesis by inhibiting oxidative DNA damage, carcinogen activation, cell proliferation, invasion, and angiogenesis. Our study suggests that the antioxidative property of bLF and P-B may be responsible for chemoprevention of HBP carcinogenesis by modulating multiple molecular targets.