MCF-7 Human Breast Cancer Xenografts Research Articles

Modeling bystander effects that cause growth delay of breast cancer xenografts in bone marrow of mice treated with radium-223

Rationale The role of radiation-induced bystander effects in cancer therapy with alpha-particle emitting radiopharmaceuticals remains unclear. With renewed interest in using alpha-particle emitters to sterilize disseminated tumor cells, micrometastases, and tumors, a better understanding of the direct effects of alpha particles and the contribution of the bystander responses they induce is needed to refine dosimetric models that help predict clinical benefit. Accordingly, this work models and quantifies the relative importance of direct effects (DE) and bystander effects (BE) in the growth delay of human breast cancer xenografts observed previously in the tibiae of mice treated with 223RaCl2. Methods A computational model of MDA-MB-231 and MCF-7 human breast cancer xenografts in the tibial bone marrow of mice administered 223RaCl2 was created. A Monte Carlo radiation transport simulation was performed to assess individual cell absorbed doses. The responses of the breast cancer cells to direct alpha particle irradiation and gamma irradiation were needed as input data for the model and were determined experimentally using a colony-forming assay and compared to the responses of preosteoblast MC3T3-E1 and osteocyte-like MLO-Y4 bone cells. Using these data, a scheme was devised to simulate the dynamic proliferation of the tumors in vivo, including DE and BE propagated from the irradiated cells. The parameters of the scheme were estimated semi-empirically to fit experimental tumor growth. Results A robust BE component, in addition to a much smaller DE component, was required to simulate the in vivo tumor proliferation. We also found that the relative biological effectiveness (RBE) for cell killing by alpha particle radiation was greater for the bone cells than the tumor cells. Conclusion This modeling study demonstrates that DE of radiation alone cannot explain experimental observations of 223RaCl2-induced growth delay of human breast cancer xenografts. Furthermore, while the mechanisms underlying BE remain unclear, the addition of a BE component to the model is necessary to provide an accurate prediction of the growth delay. More complex models are needed to further comprehend the extent and complexity of 223RaCl2-induced BE.

Protease activated receptor-1 targeted 99Tcm-(HYNIC-BMS-200261)(tricine)(TPPTS) imaging on nude mice bearing human breast cancer

Objective To study the reliability of 99Tcm-(hydrazinonicotinamide (HYNIC)-BMS-200261)(tricine)(trisodium triphenylphosphine-3, 3′, 3″-trisulfonate (TPPTS)) as a radiotracer for protease activated receptor-1 (PAR-1) expression in breast cancer. Methods Fifteen nude mice bearing MDA-MB-435, MDA-MB-231 and MCF-7 human breast cancer xenografts (5 mice for each cell line) with different PAR-1 expression were used for 99Tcm-(HYNIC-BMS-200261)(tricine)(TPPTS) γ imaging, and tumor/non-tumor (T/NT) ratios were obtained with region of interest (ROI) technique. Afterwards, the biodistribution of 99Tcm-(HYNIC-BMS-200261)(tricine)(TPPTS) was analyzed in tumor-bearing nude mice, and the radioactivity in tumor (percentage activity of injection dose per gram of tissue, %ID/g) was calculated. The immunostaining was performed to examine PAR-1 expression in tumor tissue and semi-quantitative analysis was used. One-way analysis of variance and Pearson correlation analysis were used to analyze data. Results At 2 h postinjection, the T/NT ratios were 3.03±0.32, 2.27±0.25 and 1.51±0.13 respectively in nude mice bearing MDA-MB-435, MDA-MB-231 and MCF-7 xenografts; the tumor uptakes of 99Tcm-(HYNIC-BMS-200261)(tricine)(TPPTS) were (1.03±0.15), (0.56±0.14) and (0.30±0.06) %ID/g; PAR-1 expression levels were (17.22±2.71)%, (10.78±1.95)% and (2.80±1.18)%, respectively (F values: 47.66, 46.36, 62.35, all P<0.05). The T/NT ratios and tumor uptake of 99Tcm-(HYNIC-BMS-200261)(tricine)(TPPTS) at 2 h post-injection were correlated with PAR-1 expression (r values: 0.934 and 0.929, both P<0.05). Conclusions 99Tcm-(HYNIC-BMS-200261)(tricine)(TPPTS) imaging could be a noninvasive and effective method for monitoring PAR-1 expression in human breast cancer. Key words: Breast neoplasms; Receptors, proteinase-activated; Antistatic agents; Radionuclide imaging; Tumor cells, cultured; Mice, nude

Albumin as a "Trojan Horse" for polymeric nanoconjugate transendothelial transport across tumor vasculatures for improved cancer targeting.

Although polymeric nanoconjugates (NCs) hold great promise for the treatment of cancer patients, their clinical utility has been hindered by the lack of efficient delivery of therapeutics to targeted tumor sites. Here, we describe an albumin-functionalized polymeric NC (Alb-NC) capable of crossing the endothelium barrier through a caveolae-mediated transcytosis pathway to better target cancer. The Alb-NC is prepared by nanoprecipitation of doxorubicin (Doxo) conjugates of poly(phenyl O-carboxyanhydrides) bearing aromatic albumin-binding domains followed by subsequent surface decoration of albumin. The administration of Alb-NCs into mice bearing MCF-7 human breast cancer xenografts with limited tumor vascular permeability resulted in markedly increased tumor accumulation and anti-tumor efficacy compared to their conventional counterpart PEGylated NCs (PEG-NCs). The Alb-NC provides a simple, low-cost and broadly applicable strategy to improve the cancer targeting efficiency and therapeutic effectiveness of polymeric nanomedicine.

Abstract 4897: Circadian/melatonin disruption by dim light at night drives human epithelial breast cancer to a metastatic phenotype

Abstract Cancer patients with disrupted 24-hour (circadian) rhythms are reported to have poorer survival as compared to those with normal rhythms. Severe alterations in circadian rhythms predict an increased risk of death in patients with colorectal and breast cancer, suggesting that circadian disruption may impact tumor progression and metastasis. We recently reported that circadian/melatonin (MLT) disruption by exposure to dim light at night (dLAN) resulted in constitutive phospho-activation of ERK1/2, CREB, NF-kB, and STAT3 in breast tumor xenografts promoting resistance to Tamoxifen and Doxorubicin therapy. Given that chemoresistant breast cancer is frequently metastatic, this study examined if dLAN-induced circadian/MLT disruption can promote epithelial-to- mesenchymal transition (EMT) of epithelial MCF-7 breast tumor xenografts leading to the development of metastatic foci in the lungs, livers, and brains of circadian complete (MLT-producing) athymic nude female rats and mice. Female nude rats with ERα+ MCF-7 or T47D human epithelial breast cancer xenografts were housed in LD,12:12 and LD,12:12dLAN (dLAN) photoperiods or in dLAN supplemented with nighttime MLT (0.05 µg/ml) in the drinking water, with lights on at 0600 hrs and off at 1800 hrs. Blood samples collected during the mid-dark phase (2400 hrs) showed elevated nocturnal melatonin (118.4 pg/ml) in the LD,12:12 group, but significantly suppressed melatonin (10.0 pg/ml) in the dLAN group. Tumor xenografts from rats housed in dLAN showed a ~3-fold decrease in latency-to-onset and a ~2.8-fold increase in growth rates vs. those from rats in dLAN + MLT. Tumor cAMP levels, as well as numerous signaling pathways involved in promoting EMT (Vimentin, β-catenin, and SNAIL) and metastasis (HER2/HER3, pCREB, pERK1/2, pRSK2, and pSTAT3), showed increased expression/phospho-activation at 2400 hrs in response to dLAN but repressed expression in tumors from rats in dLAN + MLT. Follow-up studies with Foxn1nude female mice implanted with non-metastatic luciferase expressing MCF-7 breast cancer cells showed that exposure to dLAN suppressed the nighttime serum levels of MLT by 93% in these mice compared to those in a LD,12:12 photoperiod. Exposure of mice to dLAN induced the rapid growth of MCF-7luc tumor xenografts and, after 5 weeks, induced the metastatic outgrowth of MCF-7 xenografts to form luciferase identifiable metastatic foci in the lungs, livers, and brains of all mice, as measured by IVIS small animal imaging system. Conversely, MCF-7luc tumor xenografts from mice exposed to dLAN and supplemented with nighttime MLT showed a reduced tumor development, 3-fold slower tumor growth, and a small metastatic lesion in one lung of a single mouse. This study is the first to show that circadian/MLT disruption by host exposure to dLAN is able to drive EMT in human epithelial breast cancer xenografts to generate metastatic foci in lung, liver, and brain of mice. Citation Format: Steven M. Hill, Shulin Xiang, Robert T. Dauchy, Melissa Wren-Dail, Murali Anbalagan, Brian Rowan, Tripp Frasch, David E. Blask. Circadian/melatonin disruption by dim light at night drives human epithelial breast cancer to a metastatic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4897. doi:10.1158/1538-7445.AM2017-4897

Abstract 3370: Metabolic profiling of the tumor interstitial fluid using 1H MRS: contribution of breast cancer subtypes and VEGF overexpression

Abstract One of the least examined, and yet critically important factors of the tumor microenvironment (TME) is the tumor interstitial fluid (TIF) that contains the tumor secretome. This fluid surrounds cancer and stromal cells and contains various cytokines, and nutritional and molecular factors that shape the outcome of nearly all aspects of tumor angiogenesis, growth, metastasis, and response to treatment. As mining of targets to treat cancer expands into the TME, the TIF represents an important source of identifying new targets in cancer treatment. Angiogenesis, one of the major hallmarks of cancer, is essential for cancers to establish vasculature for growth and hematogenous metastasis. To further understand the TIF and plasma metabolite content in breast cancer, and the role of VEGF in modifying metabolite concentrations in the TME, here we used 1H MRS to characterize metabolites in TIF collected from the triple negative MDA-MB-231 and the ER-positive MCF-7 human breast cancer xenografts with and without VEGF overexpression implanted in SCID mice. We created a collection chamber to collect IF from the tumors. The chamber was implanted with small tumor pieces in the subcutaneous space until the tumor encompassed the chamber (4 to 12 weeks depending on cell types). The tumor was then excised and the TIF and blood plasma were collected from euthanized mice. We also collected normal subcutaneous IF (SCIF) using the same chamber in healthy mice. 1H spectra showed several differences in metabolites in TIF compared to SCIF, between MDA-MB-231 and MCF-7 TIF, and with VEGF overexpression. We observed a consumption of amino acids in the TME with decreases ranging from 17% (glycine) to 77% (methionine) compared to normal SCIF. Lipids, especially polyunsaturated fatty acids, were markedly increased by VEGF overexpression in both MCF-7 and MDA-MB-231 TIF compared to the wild type TIF. Choline metabolism was also modified by VEGF overexpression and we measured increases of 69% and 20% in free choline in MDA-MB-231_VEGF and MCF-7_VEGF respectively, compared to their wild type counterpart. A stable glucose concentration was associated with a decrease in lactate (30%) and pyruvate (44%) in MDA-MB-231 tumors while an opposite pattern was observed in MDA-MB-231_VEGF TIF with decreased glucose (55%) and increased lactate (109%). Ketonic metabolism was also modified in those tumors. Beta-hydroxybutyrate and acetoacetate were higher (81% and 46% increase vs SCIF, respectively) in MDA-MB-231 TIF while acetone was increased in both VEGF-overexpressing TIF compared to the wild type (+48% for MDA-MB-231_VEGF and +44% for MCF-7_VEGF). The TIF represents an importance source of information to understand mechanisms that drive aggressiveness, and identify new targets for diagnosis and therapy of cancer. Citation Format: Louis Dore-Savard, Santosh K. Bharti, Aleksander S. Popel, Zaver M. Bhujwalla. Metabolic profiling of the tumor interstitial fluid using 1H MRS: contribution of breast cancer subtypes and VEGF overexpression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3370.

The Synthesis and Evaluations of the (6 8) Ga-Lissamine Rhodamine B (LRB) as a New Radiotracer for Imaging Tumors by Positron Emission Tomography.

Purpose. The aim of this study is to synthesize and evaluate 68Ga-labeled Lissamine Rhodamine B (LRB) as a new radiotracer for imaging MDA-MB-231 and MCF-7 cells induced tumor mice by positron emission tomography (PET). Methods. Firstly, we performed the radio synthesis and microPET imaging of 68Ga(DOTA-LRB) in athymic nude mice bearing MDA-MB-231 and MCF-7 human breast cancer xenografts. Additionally, the evaluations of 18F-fluorodeoxyglucose (FDG), as a glucose metabolism radiotracer for imaging tumors in the same xenografts, have been conducted as a comparison. Results. The radiochemical purity of 68Ga(DOTA-LRB) was >95%. MicroPET dynamic imaging revealed that the uptake of 68Ga(DOTA-LRB) was mainly in normal organs, such as kidney, heart, liver, and brain and mainly excreted from kidney. The MDA-MB-231 and MCF-7 tumors were not clearly visible in PET images at 5, 15, 30, 40, 50, and 60 min after injection of 68Ga(DOTA-LRB). The tumor uptake values of 18F-FDG were 3.79 ± 0.57 and 1.93 ± 0.48%ID/g in MDA-MB-231 and MCF-7 tumor xenografts, respectively. Conclusions. 68Ga(DOTA-LRB) can be easily synthesized with high radiochemical purity and stability; however, it may be not an ideal PET radiotracer for imaging of MDR-positive tumors.

Abstract A91: Over-the-counter melatonin supplementation in human subjects: A potentially novel chronotherapeutic approach targeting the Warburg effect and fatty acid metabolism in breast cancer therapy/prevention

Abstract Melatonin, a circadian anti-cancer hormone produced by the pineal gland during darkness at night suppresses the Warburg effect, linoleic acid (LA) uptake/metabolism and tumor cell proliferation in both estrogen receptor (ERα+) and ERα- in tissue-isolated human breast cancer xenografts. The nighttime circadian melatonin signal regulates circadian rhythms in tumor glucose and fatty acid metabolism as well as related signaling pathways that are important in controlling cell proliferative and survival mechanisms. Over-the-counter (OTC) melatonin supplements are used by millions of individuals to treat insomnia and/or jet-lag. The present study addressed the hypothesis that oral ingestion of OTC melatonin supplements by normal adult human female volunteers results in blood levels of melatonin that suppress LA uptake/metabolism, aerobic glycolysis and cell proliferative activity in human breast cancer xenografts, growing in nude female rats, directly perfused in situ with human subject donor whole-blood following melatonin intake. Twelve young, healthy premenopausal women were recruited to ingest an OTC melatonin supplement at a single dose of either 75μg, 150μg, 300μg or 1 mg during midday (low endogenous melatonin levels) resulting in low to high pharmacological blood concentrations of melatonin. A pre-supplement venous blood sample was collected from the antecubital vein of the forearm. Each subject then ingested a randomly selected oral dose of melatonin followed approximately 1 hour later by the withdrawal of a post-supplement venous blood sample. On the following day, the pre- and post-supplement whole-blood samples collected from a given subject were separately placed into a tumor perfusion reservoir. Tissue-isolated ERα- MCF-7 human breast cancer xenografts grown in female nude rats were then directly perfused in situ for 1 hour with either pre- or post-supplement oxygenated blood (37°C). Irrespective of the dose tested, melatonin induced a 50% decrease in both tumor glucose uptake and lactate release, 22% and 44% deceases in O2 uptake and CO2 production, respectively, 50% decrease in cAMP concentrations, and a 100% decrease in linoleic acid (LA) uptake and 13-hydroxyoctadecadienoic acid (13-HODE) formation. Melatonin also caused a marked decrease in the expression of phospho-AKT, GSK3β and ERK1/2, and an 85% decrease in the incorporation of [3H]thymidine into DNA. Similar results were obtained at the lowest dose of melatonin (e.g., 75μg) in ERα+ human breast cancer xenografts. The melatonin-induced suppression tumor proliferative and metabolic activity in both ERα- and ERα+ breast cancer xenografts was completely prevented by the co-perfusion with the non-selective MT1/MT22 melatonin receptor blocker S20928 consistent with the involvement of a melatonin receptor-mediated mechanism. These results indicate that oral administration of an OTC melatonin supplement at a variety of low to high doses were equally effective in suppressing the Warburg effect and key tumor proliferative and survival signaling pathways, cAMP-dependent LA uptake and metabolism to mitogenically active 13-HODE and ultimately cell proliferation in tissue-isolated human breast xenografts irrespective of ERα status via a melatonin receptor-mediated mechanism. These findings suggest that even low doses of OTC melatonin preparations may play a potentially important role as a new chronotherapeutic agent in human breast cancer treatment and/or prevention by targeting aerobic glycolysis and fatty acid signaling and metabolism. Supported by NIH Grants R21CA129875 (DEB) and R01CA54152 (SMH). Citation Format: David E. Blask, Robert T. Dauchy, Erin M. Dauchy, Steven M. Hill, Lulu Mao, Melissa M. Wren, Mary M.C. Meyaski-Schluter, Lin Yuan. Over-the-counter melatonin supplementation in human subjects: A potentially novel chronotherapeutic approach targeting the Warburg effect and fatty acid metabolism in breast cancer therapy/prevention. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A91.

Doxorubicin resistance in breast cancer is driven by light at night-induced disruption of the circadian melatonin signal.

Chemotherapeutic resistance, particularly to doxorubicin (Dox), represents a major impediment to successfully treating breast cancer and is linked to elevated tumor metabolism and tumor over-expression and/or activation of various families of receptor- and non-receptor-associated tyrosine kinases. Disruption of circadian time structure and suppression of nocturnal melatonin production by dim light exposure at night (dLEN), as occurs with shift work, and/or disturbed sleep-wake cycles, is associated with a significantly increased risk of an array of diseases, including breast cancer. Melatonin inhibits human breast cancer growth via mechanisms that include the suppression of tumor metabolism and inhibition of expression or phospho-activation of the receptor kinases AKT and ERK1/2 and various other kinases and transcription factors. We demonstrate in tissue-isolated estrogen receptor alpha-positive (ERα+) MCF-7 human breast cancer xenografts, grown in nude rats maintained on a light/dark cycle of LD 12:12 in which dLEN is present during the dark phase (suppressed endogenous nocturnal melatonin), a significant shortening of tumor latency-to-onset, increased tumor metabolism and growth, and complete intrinsic resistance to Dox therapy. Conversely, a LD 12:12 dLEN environment incorporating nocturnal melatonin replacement resulted in significantly lengthened tumor latency-to-onset, tumor regression, suppression of nighttime tumor metabolism, and kinase and transcription factor phosphorylation, while Dox sensitivity was completely restored. Melatonin acts as both a tumor metabolic inhibitor and circadian-regulated kinase inhibitor to reestablish the sensitivity of breast tumors to Dox and drive tumor regression, indicating that dLEN-induced circadian disruption of nocturnal melatonin production contributes to a complete loss of tumor sensitivity to Dox chemotherapy.

The effect of chemotherapeutic agents on tumor vasculature in subcutaneous and orthotopic human tumor xenografts.

BackgroundThe growth of solid tumors and their regrowth after treatment is dependent upon functional tumor vasculature. Some chemotherapeutic agents have shown anti-angiogenic properties but there are limited studies of the effect of chemotherapy on tumor vasculature. Here we investigate the effect of paclitaxel, 5-fluorouracil (5-FU) and doxorubicin on tumor vasculature in subcutaneous and orthotopic xenografts in mice.MethodsThe vascular density and percentage of functional blood vessels were evaluated in subcutaneous A431 human vulvar cancer xenografts, and in subcutaneous and orthotopic MCF-7 human breast cancer xenografts, following single doses of paclitaxel, 5-FU or doxorubicin.ResultsThere was no significant difference in total (CD31+) blood vessels between untreated ectopic and orthotopic MCF-7 tumors, but there was a significantly lower proportion of functional blood vessels in orthotopic tumors. After paclitaxel treatment, there was a decrease in functional tumor vasculature in A431 subcutaneous xenografts, followed by a subsequent rebound. There was a significant decrease in total vascular density on day 12 in A431 tumors following 5-FU or doxorubicin treatment, but no change in the percentage of functional vessels. An increase in functional blood vessels or percentage of functional vasculature was noted in MCF-7 subcutaneous and orthotopic xenografts following chemotherapy treatment.ConclusionsThere are differences in the vasculature and microenvironment of ectopic and orthotopic xenografts in mice. Anti-tumor effects of chemotherapy may be due, in part, to effects on tumor vasculature and may vary in different tumor models.

Inhibitory effects of Guben Yiliu Formula II (II) and its blood activation prescriptions on the growth of MCF-7 human breast cancer xenografts in nude mice.

To investigate the inhibitory effects of Guben Yiliu Formula II(II, GFII) and its blood activation prescription (BAP) on the growth of MCF-7 human breast cancer xenografts in nude mice, and explore their mechanisms of action. After the establishment of the MCF-7 human breast cancer xenograft model in nude mice, the mice in the GFII and BAP groups were administered with GFII (6.56 g/mL) and BAP (1.65 g/mL) by gavage for 28 days, respectively. The tumor volume and weight were measured twice a week throughout the treatment period. Apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The expression of microtubule associated protein 1 light chain 3 (LC3) was examined by immunohistochemistry, and Western blotting analysis was performed to detect the expression of anti-apoptotic protein Bcl-2 and LC3, as well as the effects on phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. Compared with the control group, the GFII and BAP groups could significantly inhibit the growth of MCF-7 human breast cancer xenografts in nude mice. The expression of Bcl-2 protein was lower in the GFII and BAP than in the control group, whereas both the percentage of apoptotic cells and LC3-II/LC3-I ratio were higher than in the control group. In addition, significantly reduced expression of phospho-Akt, phosphor-mTOR and mTOR were observed in the blood activation group (P<0.05). To some extent, the GFII and its BAP can exert their inhibitory effect on the growth of MCF-7 human breast cancer xenografts by inducing the cell apoptosis and autophagy. In addition to the induction of cell apoptosis, we also found that the BAP of GFII could induce cell autophagy by inhibiting of PI3K/Akt/mTOR signaling pathway, and then suppress the breast cancer cell growth.

Abstract 1047: Circadian disruption induced by light at night upregulates PCNA expression in tissue-isolated human breast cancer xenografts in nude rats

Abstract The circadian system and the cell cycle are two global regulatory systems in animals and humans. Previous studies have shown that disruption of either the circadian system or the cell cycle increases the risk of cancer in humans. However, the molecular mechanisms of circadian-mediated cell cycle dysregulation are not completely understood. Because proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and cell cycle regulation, we studied the circadian regulation and disruption of PCNA in an in vivo animal model of human breast cancer. In tissue-isolated ER- MCF-7 human breast cancer xenografts grown in female nude rats exposed to a normal 12L:12D circadian condition, PCNA protein levels were maximal in the morning (2 h after lights on) but remained at very low levels throughout the rest of the 24 h period. To determine whether circadian disruption alters PCNA protein expression, xenograft-bearing nude rats were exposed to low intensity of light at night (LAN) (0.08 μW/cm2). PCNA protein was continuously expressed at a high level throughout the 24 h period in breast cancer xenografts growing in nude rats exposed to the LAN. Exposure of tumor-bearing rats to LAN also resulted in significantly accelerated growth of these xenografts. Moreover, several signaling cascades related to cell growth were examined. Daily rhythms of Akt and MAPK activation in the human breast cancer tumors were disrupted by LAN but did not track the changes in PCNA expression; however, PDK1 activation directly correlated with PCNA expression. Expression of PKCδ and PKCα, downstream targets of PDK1, was differentially elevated by LAN in xenografts in a manner consistent with their reported roles in cell proliferation. In contrast, LAN did not disrupt the rhythmic expression of either PCNA, PKCδ, or PKCα in the liver of the tumor-bearing rats. Expression of insulin-like growth factor 1 receptor (IGF-1R) protein, an upstream signaling molecule for PDK1, also correlated with the expression pattern of PDK1/PKC/PCNA in tumor-bearing rats exposed to LAN. Exposure of tumor-bearing rats to LAN disrupted the circadian rhythm of IGF-1R protein levels in the liver. Finally, circulating IGF-1 concentrations showed circadian disruption in LAN-exposed tumor-bearing nude rats. Thus, interruption of the IGF-1 signaling pathway may constitute a novel molecular mechanism of circadian regulated tumor growth. Taken together, our results suggest that LAN-induced disruption of circadian rhythms in cell signaling cascades accelerates tumor growth in vivo through continuous up-regulation of PCNA. 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 1047.

Circadian Stage-Dependent Inhibition of Human Breast Cancer Metabolism and Growth by the Nocturnal Melatonin Signal: Consequences of Its Disruption by Light at Night in Rats and Women

The circadian production of melatonin by the pineal gland during the night provides an inhibitory signal to tissue-isolated steroid receptor SR+ and - MCF-7 human breast cancer xenografts in female nude rats. A pivotal mechanism for melatonin's anticancer effects in vivo involves a melatonin receptor-mediated inhibition of linoleic acid (LA) uptake and its metabolism to mitogenically active 13-hydroxyoctadecadienoic acid (13-HODE). Exposure of (SR-) xenograft-bearing rats to increasing intensities of polychromatic white light at night suppresses melatonin while increasing tumor growth rates, DNA content, [3H]thymidine incorporation into DNA, LA uptake, 13-HODE formation, cAMP levels and ERK1/2 activation a dose-dependent manner. Similar effects occur in SR- human breast cancer xenografts perfused in situ with melatonin-depleted blood from healthy female subjects after their exposure to a single bright intensity (2800 lux) of polychromatic light at night. Additionally, SR- human breast cancer xenografts exhibit robust circadian rhythms of LA uptake, 13-HODE formation and proliferative activity. Exposure of xenograft-bearing rats to dim light at night results in the complete elimination of these rhythms which culminates in unfettered, high rates of tumor metabolism and growth. The organization of tumor metabolism and growth within circadian time structure by the oncostatic melatonin signal helps create a balance between the cancer and its host that is disrupted by host exposure to light at night. This biological mechanism may partially explain the higher risk of breast and other cancers in women working rotating night shifts and possibly others who also experience prolonged exposure to light at night.

In vivo examination of 188Re(I)-tricarbonyl-labeled trastuzumab to target HER2-overexpressing breast cancer

Trastuzumab (Herceptin), a humanized IgG1 monoclonal antibody directed against the extracellular domain of the HER2 protein, acts as an immunotherapeutic agent for HER2-overexpressing human breast cancers. Radiolabeled trastuzumab with beta- or alpha emitters can be used as radioimmunotherapeutic agent for the similar purpose but with additional radiation effect. In this study, trastuzumab was labeled with (188)Re for radioimmunotherapy of HER2/neu-positive breast cancer. (188)Re(I)-tricarbonyl ion, [(188)Re(OH(2))(3)(CO)(3)](+), was employed as a precursor for directly labeling the monoclonal antibody with (188)Re. The immunoreactivity of (188)Re(I)-trastuzumab was estimated by competition receptor-binding assay using HER2/neu-overexpressive BT-474 human breast cancer cells. The localization properties of (188)Re(I)-trastuzumab within both tumor and normal tissues of athymic mice bearing BT-474 human breast cancer xenografts (HER2/neu-overexpressive) and similar mice bearing MCF-7 human breast cancer xenografts (HER2/neu-low expressive) were investigated. When incubated with human serum albumin and histidine at 25 degrees C, (188)Re(I)-trastuzumab was found to be stable within 24 h. The IC(50) of (188)Re(I)-trastuzumab was found to be 22.63+/-4.57 nM. (188)Re(I)-trastuzumab was shown to accumulate specifically in BT-474 tumor tissue in in vivo biodistribution studies. By microSPECT/CT, the image of (188)Re localized BT-474 tumor was clearly visualized within 24 h. In contrast, (188)Re(I)-trastuzumab uptake in HER2-low-expressing MCF-7 tumor was minimal, and the (188)Re image at the localization of the tumor was dim. These results reveal that (188)Re(I)-trastuzumab could be an appropriate radioimmunotherapeutic agent for the treatment of HER2/neu-overexpressing cancers.

A Parenteral Econazole Formulation Using a Novel Micelle-to-Liposome Transfer Method: In Vitro Characterization and Tumor Growth Delay in a Breast Cancer Xenograft Model

The purpose of this study was to develop a parenteral liposomal formulation of econazole, a poorly water-soluble compound not previously available in an intravenous form. We are investigating econazole as an anticancer agent based on its unique mechanism of action to which cancer cells are preferentially sensitive. An intravenous formulation of econazole was desired for preclinical toxicity and efficacy studies of econazole. Liposomal econazole was prepared using a novel micelle exchange technique to incorporate the drug into the lipid bilayer of pre-formed liposomes using a poly(ethylene) glycol-linked phospholipid, distearoyl phosphatidylethanolamine (DSPE-PEG). This method allowed for stable and efficient drug incorporation into DPPC and DMPC liposomes at a final drug:lipid ratio of 0.05 (w/w) and increased solubility in saline from <0.1 to 5 mg/ml. Stability over 14 days at 4 degrees C in buffer was demonstrated as well as in vitro plasma stability at 37 degrees C. Plasma elimination studies of micelle-loaded liposomal econazole showed a half-life of approximately 35 min and plasma AUC of 281 microg/ml min. In MCF-7 human breast cancer xenografts in Rag2M mice. Liposomal econazole did not induce significant hepatoxicity, renal toxity or weight loss compared to empty liposomes. Tumor growth was slightly delayed in liposomal econazole-treated mice, with approximately 10-day lag time to reach 300 mm(3) compared to vehicle controls. The micelle transfer method provided an efficient means of preparing liposomal econazole suitable for intravenous administration. Liposomal econazole was successfully administered to tumor bearing mice at 50 mg/kg, and no significant toxicities attributable to econazole were observed.

Mammalian lignans enterolactone and enterodiol, alone and in combination with the isoflavone genistein, do not promote the growth of MCF‐7 xenografts in ovariectomized athymic nude mice

This study determined the effect of the mammalian lignans enterolactone (ENL) and enterodiol (END) alone and in combination with the isoflavone genistein (GEN) on the growth of MCF-7 tumors in ovariectomized nude mice. Ovariectomized athymic nude mice with established MCF-7 tumors were fed a basal diet (AIN-93G) and divided into 5 groups that received daily subcutaneous injections (10 mg/kg body weight (BW)) of ENL, END, GEN, a mixture of these compounds (MIX), or vehicle as a negative control for 22 weeks. A positive control group was implanted with an estradiol pellet in order to establish an estrogenic tumor growth response. In the ENL- and END-treated mice, palpable tumors regressed significantly by 91 and 83%, respectively, resulting in final tumors that were similar to the negative control tumors. However, tumor cell apoptosis was significantly enhanced by the lignans. In the GEN-treated mice, tumors initially regressed significantly by 64% but regression ceased following prolonged treatment, resulting in final tumors that were significantly larger compared to negative control, ENL-, and END-treated mice, in part by increasing tumor cell proliferation. The MIX treatment significantly regressed palpable tumors by 87% similar to negative control group, with no effects on tumor cell apoptosis or proliferation. The isoflavone GEN alone promoted the growth of established MCF-7 human breast cancer xenografts after prolonged treatment while the mammalian lignans ENL and END did not. When these phytoestrogens were given in combination, no tumor growth-promoting effects were observed. (c) 2005 Wiley-Liss, Inc.

Eicosapentaenoic Acid Suppresses Cell Proliferation in MCF-7 Human Breast Cancer Xenografts in Nude Rats via a Pertussis Toxin–Sensitive Signal Transduction Pathway

The type and content of dietary PUFAs have profound influences on the growth rate of transplantable human breast cancers in immunodeficient rodents. Diets enriched in linoleic acid (LA), an (n-6) fatty acid, stimulate tumor growth, whereas dietary fats containing (n-3) fatty acids slow such growth. Interactions between LA and (n-3) fatty acids capable of regulating cell proliferation in solid tumors in vivo are not yet well defined. Here we tested the hypothesis that plasma eicosapentaenoic acid (EPA), an (n-3) fatty acid, suppresses cell proliferation in MCF-7 human breast cancer xenografts via a pertussis toxin-sensitive reduction of intratumor cAMP, LA uptake, and formation of the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) from LA. Plasma fatty acid uptake and 13-HODE release were determined in control and EPA-treated xenografts from arteriovenous differences measured during perfusion in situ. Intratumor cAMP, extracellular signal-regulated kinase p44/p42 (ERK1/2) phosphorylation, and [3H]thymidine incorporation (TTI) were measured in tumors freeze-clamped at the end of the perfusions. Arterial blood containing EPA caused significant decreases (P < 0.05) in cAMP, uptake of SFA, monounsaturated fatty acids, and (n-6) PUFA, 13-HODE formation, ERK1/2 phosphorylation, and TTI in MCF-7 xenografts. These effects of EPA were reversed by the addition of either pertussis toxin or 8-bromoadenosine-cAMP to the EPA-containing arterial blood. Addition of 13-HODE to the EPA-containing arterial blood restored phosphorylated ERK1/2 and TTI but not FA uptake. The results suggest that EPA regulates cell proliferation in MCF-7 xenografts via a novel inhibitory G protein-coupled, (n-3) FFA receptor-mediated signal transduction pathway.

131I-recombinant human EGF has antitumor effects against MCF-7 human breast cancer xenografts with low levels of EGFR

This study investigated the inhibitory action of 131I-recombinant human EGF ( 131I-rhEGF) on MCF-7 human breast cancer tumor development in nude mice. The activity and tumor uptake of 131I-rhEGF was measured by tissue distribution assay, and its effect on tumor growth was measured by monitoring tumor size after treatment with 131I-rhEGF. Changes in tumor cell ultrastructure were observed by transmission electron microscopy (TEM), and pathological changes in tumor tissue were observed by light microscopy. The tissue distribution assay revealed that 131I-rhEGF was markedly absorbed by the tumor and reached its maximal uptake rate (16.73%ID · g −1) at 120 hours at which point the drug concentration in the tumor was 11.1-fold, 8.1-fold, and 6.6-fold higher than that in blood, liver, and kidneys, respectively. Tumor size measurements showed that tumor development was significantly inhibited by intravenously and intratumorally injected 131I-rhEGF. Tumor inhibition rates (82.0% and 80.7%, respectively) were significantly higher than those of tumors treated with 131I (7.49%) and 131I-HSA (6.91%; P < 0.05). TEM and light microscopy revealed that intravenous and intratumoral injection of 131I-rhEGF could significantly damage and ultimately kill tumor cells. Our results suggest that 131I-rhEGF suppresses development of xenografted breast cancer cells in nude mice, providing a novel candidate for receptor-mediated targeted radiotherapy.

Glycolysis and glucose transporter 1 as markers of response to hormonal therapy in breast cancer.

Estrogen plays a key role in the development and progression of breast cancer; hence, antiestrogens, such as tamoxifen, have a marked impact on the treatment and outcome of breast cancer patients. Estrogen-induced growth requires continuous replenishment of energy, predominantly generated by glycolysis. Previous work from this laboratory demonstrated estrogen induction and tamoxifen inhibition of glycolysis in MCF7 human breast cancer cells in vitro (Furman et al., J Steroid Biochem Mol Biol 1992;43:189-95). We present here studies of estrogen vs. tamoxifen regulation of glycolysis in orthotopic MCF7 human breast cancer xenografts in vivo. In addition we investigated mediation of this metabolic regulation through glucose transporter 1, in the same cells, in vitro, as well as in 2 other hormone-responsive human breast cancer cells. Tumor response and glycolysis were monitored noninvasively by means of magnetic resonance imaging and 13C spectroscopy, respectively. During estrogen-stimulated tumor growth (from approximately 0.5 to approximately 1.3 cm3 in 10 days), the rate of glucose metabolism through glycolysis in vivo was high at 40 +/- 4 micromole/g/min. However, treatment for 10 days with tamoxifen induced growth arrest and a concomitant decrease of 2-fold in the rate of glycolysis. In congruence, glucose transporter 1 expression was stimulated by estrogen, reaching after 72 hr a 2- to 3-fold higher level of expression relative to that in tamoxifen-treated cells. Thus, estrogen-induced changes in glycolysis appeared to be mediated via its regulation of glucose transporter 1 expression. The in vivo monitoring of glycolysis may serve as a tool to expose hormonal regulation of glucose transporter 1 expression in breast cancer tumors, as well as to assess response to hormonal therapy.

Growth and fatty acid metabolism of human breast cancer (MCF-7) xenografts in nude rats: impact of constant light-induced nocturnal melatonin suppression.

The nocturnal melatonin (MLT) surge is a relevant oncostatic signal for a variety of experimental malignancies. Population studies support the hypothesis that exposure to light at night may represent a new risk factor for breast cancer possibly through the suppression of pineal MLT production and/or circadian disruption. We tested the ability of constant light exposure to suppress MLT production in female nude rats and stimulate the growth of tissue-isolated MCF-7 human breast cancer xenografts via increased tumor linoleic acid (LA) metabolism. Rats maintained on an alternating light/dark cycle (L:D group) exhibited a robust circadian MLT rhythm that was abolished following constant light exposure. During the exposure of animals bearing tissue-isolated human MCF-7 breast cancer xenografts to constant light, the rate of tumor growth markedly increased relative to the L:D group. Tumor LA uptake and its metabolism to the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) were also substantially higher under constant light conditions. This is the first biological evidence for a potential link between constant light exposure and increased human breast oncogenesis involving MLT suppression and stimulation of tumor LA metabolism.

Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles.

Unmethylated CpG dinucleotides present within certain specific sequence contexts in bacterial and synthetic DNA stimulate innate immune responses and induce cytokine secretion. Recently, we showed that CpG DNAs containing two 5'-ends, immunomers, are more potent in both regards. In this study, we show that an immunomer containing a synthetic CpR motif (R = 2'-deoxy-7-deazaguanosine) is a potent immunostimulatory agent. However, the profile of cytokine induction is different from that with immunomers containing a natural CpG motif. In general, a CpR immunomer induced higher interleukin (IL)-12 and lower IL-6 secretion. Compared with conventional CpG DNAs, both types of immunomers showed a rapid and enhanced activation of the transcription factor NF-kappaB in J774 cells. NF-kappaB activation by CpG DNA corresponded to degradation of IkappaBalpha in J774 cells. All three immunostimulatory oligonucleotides activated the p38 mitogen-activated protein kinase pathway as expected. Immunomers containing CpG and CpR motifs showed potent reversal of the antigen-induced Th2 immune response towards a Th1 type in antigen-sensitized mouse spleen cell cultures. Immunomers containing a CpR motif showed significant antitumor activity in nude mice bearing MCF-7 human breast cancer and U87MG glioblastoma xenografts. These studies suggest the ability for a divergent synthetic nucleotide motif recognition pattern of the receptor involved in the immunostimulatory pathway and the possibility of using synthetic nucleotides to elicit different cytokine response patterns.