Effects of Selenium and Low Levels of Lead on Mammary Tumor Development and Growth in MMTV-infected Female Mice

Selenium (Se) has been demonstrated in previous studies to inhibit mammary tumorigenesis in C3H mice infected with the murine mammary tumorvirus, MMTV. The antitumorigenic effects of Se in this animal model of breast cancer were subsequently shown to be counteracted by Se-antagonistic elements. Lead (Pb), for example, was found to abolish the anticarcinogenic effects of Se at 5 ppm in the drinking water. The present study was undertaken to explore the effects of Pb at just 0.5 ppm in the water, i.e., at a level comparable to the concentrations of Pb that have been measured in the tap water of older homes in some communities. Groups of 30 female virgin C3H/St mice infected with MMTV maintained on Torula yeast-based diets containing either 0.15 or 0.65 ppm of yeast-based organic Se and received either deionized water or water containing 0.5 ppm Pb as the acetate over their entire postweaning lifespan. In the control group on deionized water and the 0.15 ppm Se feed, the tumor incidence was 78.6%, which is normal for this strain. Increasing the Se content of the feed to 0.65 ppm lowered the tumor incidence to 30%, demonstrating the antitumorigenic effect of Se. In the experimental groups, the Pb-exposed mice on the 0.15 ppm Se feed developed signs of chronic Pb toxicity as evidenced by diminished weight gain that persisted up to the age of 10 months, during which period the animals remained tumor-free. Thereafter, weight gains ensued to near the values of the controls, and the tumors began to develop in rapid succession until the final tumor incidence of 73.7% was reached. In the group of mice on the 0.65 ppm Se feed, the toxic effects of Pb were diminished, as evidenced by the normal weight gains during the first 10 months but with concomitant physiological inactivation of Se, causing 82.6% of the mice to develop tumors, with the first tumor to appear at the age of 5 months, 7 months earlier than in the Pb-unexposed controls. In addition, tumor growth rates in this group were greatly accelerated and the survival of the tumor-bearing animals was significantly shortened. Direct evidence for the interactions of Pb with Se were obtained by determinations of the two elements in the livers, kidneys, and hair of tumor-free and tumor-bearing mice. However, the exposure of the mice to Pb in the water also altered the levels of Zn, Cu, Fe, and Cr in the organs and tissues, more so in tumor-bearing than tumor-free animals. The present study demonstrates the need to consider the interactions of Se with other trace elements in discussions of its mechanism of anticarcinogenic action.     

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.     

Mammary Gland Growth Factors: Roles in Normal Development and in Cancer

Normal development of the mammary gland proceeds via interactions between the epithelium and the mesenchyme that start during embryogenesis and continue during pubertal outgrowth and differentiation. The function of specific peptide growth factors that bind members of the receptor tyrosine kinase family and the cytokine receptor family are required at each stage. In many cases the peptides are produced in one compartment and act on receptors in the other compartment. One of the striking differences between normal development and cancer is the loss of this cross-talk. Mammary tumor cells often produce a peptide and express the receptor on the same cell leading to autocrine activation of signaling pathways, a mechanism that is characteristic for cancer cells. We will discuss different peptides in the context of normal development and cancer in this review.Development of the mouse mammary gland begins at embryonic day 10.5 with formation of the milk line, a thickening of the ectoderm that extends from the anterior to the posterior limb buds. By E11.5 five placode pairs invaginate into the surrounding stroma and undergo limited branching morphogenesis. At birth a rudimentary ductal tree is present in a stromal fat pad. At the onset of puberty ovarian hormones induce ductal elongation until the fat pad is filled with a system of ducts and side branches. During pregnancy the glands fill with secretory alveolar units that on parturition produce copius amounts of milk for the offspring. As the pups are weaned, these alveoli are removed by programmed cell death coupled with extensive tissue remodeling, an event termed involution. All these processes are under the control of a wide variety of hormonal and peptide factors. In this review, which is concentrated on peptide growth factors, we will discuss those factors that have important roles in normal development and have been implicated in breast cancer.     

siRNA-Based Targeting of Cyclin E Overexpression Inhibits Breast Cancer Cell Growth and Suppresses Tumor Development in Breast Cancer Mouse Model

Cyclin E is aberrantly expressed in many types of cancer including breast cancer. High levels of the full length as well as the low molecular weight isoforms of cyclin E are associated with poor prognosis of breast cancer patients. Notably, cyclin E overexpression is also correlated with triple-negative basal-like breast cancers, which lack specific therapeutic targets. In this study, we used siRNA to target cyclin E overexpression and assessed its ability to suppress breast cancer growth in nude mice. Our results revealed that cyclin E siRNA could effectively inhibit overexpression of both full length and low molecular weight isoforms of cyclin E. We found that depletion of cyclin E promoted apoptosis of cyclin E-overexpressing cells and blocked their proliferation and transformation phenotypes. Significantly, we further demonstrated that administration of cyclin E siRNA could inhibit breast tumor growth in nude mice. In addition, we found that cyclin E siRNA synergistically enhanced the cell killing effects of doxorubicin in cell culture and this combination greatly suppressed the tumor growth in mice. In conclusion, our results indicate that cyclin E, which is overexpressed in 30% of breast cancer, may serve as a novel and effective therapeutic target. More importantly, our study clearly demonstrates a very promising therapeutic potential of cyclin E siRNA for treating the cyclin E-overexpressing breast cancers, including the very malignant triple-negative breast cancers.     

两种人乳腺癌裸鼠移植模型的建立

目的建立简便的人乳腺癌裸鼠移植模型,并探讨其部分生物学特性。方法采用雌激素受体阴性的MDA-MB-231和SK-BR-3人乳腺癌细胞株,分别接种于10只裸鼠左侧腋窝皮下,移植细胞总数为1×107/只。观察肿块生长情况,第42天处死荷瘤鼠,切除肿块作病理切片。结果 MDA-MB-231接种后第5d在接种部位可见结节,成瘤率为90%（9/10）,接种42 d肿瘤体积426.6±333.8,瘤重0.417±0.276,病理学检查为浸润性导管癌;SK-BR-3接种后第11天在接种部位可见结节,成瘤率为80%（8/10）,接种42 d肿瘤体积357.5±246,瘤重0.325±0.167,病理学检查为浸润性导管癌。结论该方法建立的人乳腺癌裸鼠移植模型,皮下移植方法简单,易于操作,成功率较高,肿瘤可部分保持人乳腺癌生物学特性,为研究人乳腺癌提供了重要工具。     

Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer

As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.     

The Major Pre- and Postmenopausal Estrogens Play Opposing Roles in Obesity-Driven Mammary Inflammation and Breast Cancer Development

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Potential Effects of Mina53 on Tumor Growth in Human Pancreatic Cancer

Myc-induced nuclear antigen (Mina53) is a protein with a molecular weight of 53 kDa expression of which is induced by c-Myc. Increased expression of Mina53 is documented in some human carcinomas. In this study, we found markedly increased Mina53 expression in pancreatic cancer tissue specimens. This expression did not correlate with clinicopathological characteristics, such as sex, age, and presence of distant metastasis. However, there was a statistically significant association with histological differentiation, TNM stage, and lymph node metastases. To study functional role of Mina53, we silenced its expression by siRNA in PANC-1 cells. These cells were arrested in the G2/M phase, and apoptosis rates were increased. In conclusion, increased expression of Mina53 may play an important role in the development of human pancreatic cancer. Mina53 can be used as a marker for pancreatic cancer and may potentially be exploited as a target for treatment of pancreatic cancer.     

人卵巢癌裸鼠移植实体瘤模型的建立

目的建立人卵巢癌裸鼠移植实体瘤模型。方法将1例人卵巢癌组织移植裸鼠,建立人卵巢癌裸鼠原代移植实体瘤模型的基础上,再将实体瘤皮下移植、实体瘤原位移植、实体瘤细胞移植裸鼠。观察裸鼠实体瘤生长和转移情况,称量其体重、子宫卵巢重、瘤重及瘤的大小,并作病理、电镜、染色体检查。结果成功地建立人卵巢癌裸鼠移植实体瘤模型,并已传至第18代,传代移植成功率100％,组织学和超微结构形态均证明该实体瘤保留了原人卵巢癌特征,有人卵巢癌染色体特征,并出现肝、脾转移。结论本研究建立人卵巢癌裸鼠移植实体瘤模型与人相似,通过18代传代和实验观察方法稳定可靠。为人卵巢癌的研究提供了理想的动物模型。     

A New Mouse Model for the Study of Human Breast Cancer Metastasis

Breast cancer is the most common cancer in women, and this prevalence has a major impact on health worldwide. Localized breast cancer has an excellent prognosis, with a 5-year relative survival rate of 85%. However, the survival rate drops to only 23% for women with distant metastases. To date, the study of breast cancer metastasis has been hampered by a lack of reliable metastatic models. Here we describe a novel in vivo model using human breast cancer xenografts in NOD scid gamma (NSG) mice; in this model human breast cancer cells reliably metastasize to distant organs from primary tumors grown within the mammary fat pad. This model enables the study of the entire metastatic process from the proper anatomical site, providing an important new approach to examine the mechanisms underlying breast cancer metastasis. We used this model to identify gene expression changes that occur at metastatic sites relative to the primary mammary fat pad tumor. By comparing multiple metastatic sites and independent cell lines, we have identified several gene expression changes that may be important for tumor growth at distant sites.     

转基因小鼠乳腺表达人瘦蛋白的研究

利用转基因动物乳腺生产药用蛋白质是近年来研究的热点,在这方面已有不少成功的例子,展现出良好的应用前景[1,2].本研究选择人瘦蛋白基因作为目标基因是因为其表达产物瘦蛋白能对人体内脂肪的蓄积和能量消耗进行有效的反馈调控,美国科学家已将用E.coli表达的人瘦蛋白用于人肥胖症的治疗并取得了良好的治疗效果[3],但尚未见到利用转基因动物乳腺表达这种蛋白质的研究报道.     

A Novel 3-D Mineralized Tumor Model to Study Breast Cancer Bone Metastasis

Background

Metastatic bone disease is a frequent cause of morbidity in patients with advanced breast cancer, but the role of the bone mineral hydroxyapatite (HA) in this process remains unclear. We have developed a novel mineralized 3-D tumor model and have employed this culture system to systematically investigate the pro-metastatic role of HA under physiologically relevant conditions in vitro.

Methodology/Principal Findings

MDA-MB231 breast cancer cells were cultured within non-mineralized or mineralized polymeric scaffolds fabricated by a gas foaming-particulate leaching technique. Tumor cell adhesion, proliferation, and secretion of pro-osteoclastic interleukin-8 (IL-8) was increased in mineralized tumor models as compared to non-mineralized tumor models, and IL-8 secretion was more pronounced for bone-specific MDA-MB231 subpopulations relative to lung-specific breast cancer cells. These differences were pathologically significant as conditioned media collected from mineralized tumor models promoted osteoclastogenesis in an IL-8 dependent manner. Finally, drug testing and signaling studies with transforming growth factor beta (TGFβ) confirmed the clinical relevance of our culture system and revealed that breast cancer cell behavior is broadly affected by HA.

Conclusions/Significance

Our results indicate that HA promotes features associated with the neoplastic and metastatic growth of breast carcinoma cells in bone and that IL-8 may play an important role in this process. The developed mineralized tumor models may help to reveal the underlying cellular and molecular mechanisms that may ultimately enable more efficacious therapy of patients with advanced breast cancer.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

Survival in Human Breast Cancer: Effects of Tumor Laterality and the Time of Year of Surgery

In a previous study, the content of sex hormone receptors in breast cancer tissue was found to be significantly correlated with tumor laterality and the time of year of surgery. The present study extended these observations by demonstrating that the five-year survival rate was also correlated with these factors.     

Connexins and Gap Junctions in Mammary Gland Development and Breast Cancer Progression

The development and function of the mammary gland require precise control of gap junctional intercellular communication (GJIC). Here, we review the expression and function of gap junction proteins, connexins, in the normal mouse and human mammary gland. We then discuss the possible tumor-suppressive role of Cx26 and Cx43 in primary breast tumors and through the various stages of breast cancer metastasis and consider whether connexins or GJIC may actually promote tumorigenesis at some stages. Finally, we present in vitro data on the impact of connexin expression on breast cancer cell metastasis to the bone. We observed that Cx43 expression inhibited the invasive and migratory potentials of MDA-MB-231 breast cancer cells in a bone microenvironment, provided by the MC3T3-E1 mouse osteoblastic cell line. Expression of either Cx26 or Cx43 had no effect on MDA-MB-231 growth and adhesion under the influence of osteoblasts and did not result in regulation of osteogenic gene expression in these breast cancer cells. Furthermore, connexin-expressing MDA-MB-231 cells did not have an effect on the growth or differentiation of MC3T3-E1 cells. In summary, we conclude that connexin expression and GJIC are integral to the development and differentiation of the mammary gland. In breast cancer, connexins generally act as tumor suppressors in the primary tumor; however, in advanced breast tumors, connexins appear to act as both context-dependent tumor suppressors and facilitators of disease progression.     

An Antigen in Human Breast Cancer Sera related to the Murine Mammary Tumour Virus

THE possibility of an oncogenic virus in human breast cancer has been increased by recent findings. Virus-like particles resembling the mammary tumour virus (MTV) were found in electron micrographs of human breast cancer tissue¹ and particles physically and morphologically similar to MTV particles have been seen in human milk². These particles were found more frequently in the milk of American women with a history of breast cancer in their immediate families and in the milk of Parsi women in Bombay than in the milk of nonselected American women³. Parsi women are three times more likely to have breast cancer than other women in Bombay³. The detection of RNA-dependent DNA-polymer-ase activity in such particles isolated from human milk emphasized the possibility that these particles represent an oncogenic RNA virus⁴. In addition to the physical and morphological resemblance of human milk particles and MTV an immunological relationship between these two kinds of particles seems probable; sera from breast cancer patients neutralize the biological activity of MTV whereas normal human sera did not do so⁵. We report data supporting the hypothesis of an immunological cross relationship between MTV and human breast cancer.     

Angiopoietin 1 Promotes Tumor Angiogenesis and Tumor Vessel Plasticity of Human Cervical Cancer in Mice

Angiopoietins have been increasingly implicated to play important roles in blood vessel formation, remodeling, maturation, and maintenance. However, their roles in tumor angiogenesis and hence tumor growth and metastasis still remain uncertain. In this work, angiopoietin 1 expression was amplified in human cervical cancer HeLa cells by stable transfection or recombinant human adenovirus-mediated gene transfer. We show that increased angiopoietin 1 expression promoted in vivo growth of human cervical cancers in mice by promoting tumor angiogenesis and inhibiting tumor cell apoptosis. Furthermore, we also show for the first time that overexpression of angiopoietin 1 also leads to increased tumor vessel plasticity with a large number of vessels lacking periendothelial supporting cells. These results indicate that angiopoietin 1 promotes tumor angiogenesis and tumor vessel plasticity of human cervical cancer in mice.     

A Probable Risk Factor of Female Breast Cancer: Study on Benign and Malignant Breast Tissue Samples

The study reports enhanced Fe, Cu, and Zn contents in breast tissues, a probable risk factor of breast cancer in females. Forty-one formalin-fixed breast tissues were analyzed using atomic absorption spectrophotometry. Twenty malignant, six adjacent to malignant and 15 benign tissues samples were investigated. The malignant tissues samples were of grade 11 and type invasive ductal carcinoma. The quantitative comparison between the elemental levels measured in the two types of specimen (benign and malignant) tissues (removed after surgery) suggests significant elevation of these metals (Fe, Cu, and Zn) in the malignant tissue. The specimens were collected just after mastectomy of women aged 19 to 59 years from the hospitals of Islamabad and Rawalpindi, Pakistan. Most of the patients belong to urban areas of Pakistan. Findings of study depict that these elements have a promising role in the initiation and development of carcinoma as consistent pattern of elevation for Fe, Cu, and Zn was observed. The results showed the excessive accumulation of Fe (229?±?121 mg/L) in malignant breast tissue samples of patients (p?<?0.05) to that in benign tissues samples (49.1?±?11.4 mg/L). Findings indicated that excess accumulation of iron in malignant tissues can be a risk factor of breast cancer. In order to validate our method of analysis, certified reference material muscle tissue lyophilized (IAEA) MA-M-2/TM was analyzed for metal studied. Determined concentrations were quite in good agreement with certified levels. Asymmetric concentration distribution for Fe, Cu, and Zn was observed in both malignant and benign tissue samples.