Kathleen F. Arcaro

The Role of Surface Functionality on Acute Cytotoxicity, ROS Generation and DNA Damage by Cationic Gold Nanoparticles

Gold nanoparticles (AuNPs) are promising materials for biomedical applications [1,2] due to their tunable surface properties [3] and extraordinary stability.[4] Additionally, the inert core material reduces the potential for toxicity issues arising from particle degradation.[5] The size regime and concomitant geometrical outcomes including high degree of curvature, however, generates the potential for toxicity.[6,7] Generally, the toxicity of AuNPs depends on size, shape, the degree to which they aggregate, and their surface properties[8,9] Recently, several studies on the short-term cytotoxicity of AuNPs[10] and quantum dots[11] have focused on size,[12,13] shape,[14,15] and charge.[16] To date, however, issues such as ligand hydrophobicity have not been systematically explored.

Surface Properties Dictate Uptake, Distribution, Excretion, and Toxicity of Nanoparticles in Fish

There are currently more than 1000 consumer products based on nanomaterials.[1] As a consequence, nanomaterials will inevitably be released into the environment during the manufacture, use, and disposal of these products. Consequently, multiple concerns related to unforeseen health and environmental hazards of nanomaterials have been raised.[2] Many studies on the environmental fate, behavior, bioavailability and toxicity of manufactured nanomaterials have been carried out to address what these hazards might be, but the results are far from conclusive.[3] Currently, carbon-based materials (e.g. fullerenes[4] and carbon nanotubes[4c, 5]), metal and metal oxides (e.g. silver[6] and TiO2[7]) and semiconductor materials (e.g. quantum dots[8]) are the most studied nanomaterials. Investigations show that nanomaterial size,[6, 9] shape,[9] surface chemistry,[4b, 10] and surface area[11] all play a role in determining the toxicity of nanomaterials in model biological and environmental systems.[3a] For example, Chan et al.[9] has demonstrated the size dependence of cellular uptake, and our group[10b] has shown the role of surface charge on uptake.

Determination of free Bisphenol A (BPA) concentrations in breast milk of U.S. women using a sensitive LC/MS/MS method

Bisphenol A (BPA) is a synthetic, endocrine-disrupting compound. Free BPA has been detected in human samples indicating that humans are internally exposed to estrogenically active BPA. The purpose of this study was to develop a sensitive method to detect free BPA in human breast milk. BPA was isolated from the milk of 21 nursing mothers in the U.S. by solid-phase extraction. It was then derivatized to improve sensitivity and subsequently analyzed by ultra high performance liquid chromatography-tandem mass spectrometry. Free BPA was detected in 62% of the milk samples (≤ 0.22-10.8 ng mL(-1), median 0.68 ng mL(-1), mean 3.13 ng mL(-1)). No statistical difference in BPA concentrations was observed between women with a low or high Body Mass Index (BMI) (<30 (n=11) and>30 (n=10), respectively). However, there was a significant association between BPA concentration and race. Caucasian women had significantly higher levels of free BPA in their breast milk than non-Caucasian women (mean=4.44 (n=14) and 0.52 (n=7), respectively; p<0.05). The difference between races could be attributed to variations in exposure, lifestyle or metabolism and suggests that certain populations should take extra precautions to limit BPA exposure, particularly during pregnancy and lactation.

Use of an Aggressive MCF-7 Cell Line Variant, TMX2-28, to Study Cell Invasion in Breast Cancer

An estrogen receptor–negative variant of the MCF-7 breast cancer cell line, TMX2-28, was used as a model in which to study breast cancer cell invasion. Using a reconstituted basement membrane (Matrigel) assay to evaluate cell invasion, we determined that TMX2-28 cells are more invasive than MCF-7 cells and that the invasiveness of TMX2-28 is similar to that of the aggressive MDA-MB-231 breast cancer cell line. TMX2-28 cells displayed a rounded, epithelial cell–like morphology, suggesting an amoeboid mode of cell invasion, in contrast to the mesenchymal mode of invasion characteristic of spindle-shaped, fibroblast-like MDA-MB-231 cells. Using real-time reverse transcription-PCR, we found that mitogen-inducible gene 2 (MIG2) is expressed at a 17-fold higher level in TMX2-28 cells than in nonaggressive MCF-7 cells and that MIG2 mRNA levels are low in the nontumorigenic human mammary epithelial cell line, 184. We determined that MIG2 plays a role in cell invasion by using small interfering RNA (siRNA) to suppress the expression of MIG2 mRNA levels in TMX2-28 cells. TMX2-28 cell invasion was reduced by 48% when the cells were transfected with siRNAs targeting MIG2, relative to cells transfected with siRNAs against glyceraldehyde-3-phosphate dehydrogenase. Finally, MIG2 expression was evaluated in reductive mammoplasty and breast tumor tissue. Although all 21 normal tissues from reduction mammoplasty showed immunoreactivity for MIG2, ranging from weak (62%) to strong (24%), only half of the 34 formalin-fixed breast tumors showed immunoreactivity for MIG2. Of these 17 positive cases, 10 were considered to overexpress MIG2 (moderate to strong staining). Examination of 30 frozen breast tumors supported the finding that MIG2 is overexpressed in a subset of breast cancers. We suggest that MIG2s normal regulation and function are disrupted in breast cancer. (Mol Cancer Res 2006;4(12):905–13)

Postpartum remodeling, lactation, and breast cancer risk: summary of a National Cancer Institute-sponsored workshop.

The pregnancy-lactation cycle (PLC) is a period in which the breast is transformed from a less-developed, nonfunctional organ into a mature, milk-producing gland that has evolved to meet the nutritional, developmental, and immune protection needs of the newborn. Cessation of lactation initiates a process whereby the breast reverts to a resting state until the next pregnancy. Changes during this period permanently alter the morphology and molecular characteristics of the breast (molecular histology) and produce important, yet poorly understood, effects on breast cancer risk. To provide a state-of-the-science summary of this topic, the National Cancer Institute invited a multidisciplinary group of experts to participate in a workshop in Rockville, Maryland, on March 2, 2012. Topics discussed included: 1) the epidemiology of the PLC in relation to breast cancer risk, 2) breast milk as a biospecimen for molecular epidemiological and translational research, and 3) use of animal models to gain mechanistic insights into the effects of the PLC on breast carcinogenesis. This report summarizes conclusions of the workshop, proposes avenues for future research on the PLC and its relationship with breast cancer risk, and identifies opportunities to translate this knowledge to improve breast cancer outcomes.

Detection of promoter methylation of tumor suppressor genes in serum DNA of breast cancer cases and benign breast disease controls

Tumors are capable of shedding DNA into the blood stream. This shed DNA may be recovered from serum or plasma. The objective of this study was to evaluate whether pyrosequencing promoter DNA in a panel of 12 breast cancer-related genes (APC, BRCA1, CCND2, CDH1, ESR1, GSTP1, HIN1, P16, RARβ, RASSF1, SFRP1 and TWIST) to measure the degree of methylation would lead to a useful serum-based marker of breast cancer. Serum was obtained from women who were about to undergo a breast biopsy or mastectomy at three hospitals from 1977 to 1987 in Grand Rapids, MI USA. We compared the methylation status of 12 genes in serum DNA obtained from three groups of postmenopausal women (mean age at blood collection: 63.0 y; SD 9.9; range 35–91): breast cancer cases with lymph node-positive disease (n = 241); breast cancer cases with lymph node-negative disease (n = 63); and benign breast disease control subjects (n = 234). Overall, median levels of promoter methylation were low, typically below 5%, for all genes in all study groups. For all genes, median levels of methylation were higher (by 3.3 to 47.6%) in lymph node-positive breast cancer cases than in the controls. Comparing mean methylation level between lymph-node positive cases and controls, the most statistically significant findings, after adjustment of the false-positive rate (q-value), were for TWIST (p = 0.04), SFRP1 (p = 0.16), ESR1 (p = 0.17), P16 (p = 0.19) and APC (p = 0.19). For two of these four genes (TWIST, P16), the median methylation level was also highest in lymph-node positive cases, intermediate in lymph node-negative cases and lowest in the controls. The percent of study subjects with mean methylation scores ≥ 5% was higher among lymph node-positive cases than controls for ten genes, and significantly higher for HIN1 and TWIST (22.0 vs. 12.2%, p = 0.04 and 37.9 vs. 24.5%, p = 0.004, respectively). Despite relatively consistent variation in methylation patterns among groups, these modest differences did not provide sufficient ability to distinguish between cases and controls in a clinical setting.

PLD1 is overexpressed in an ER-negative MCF-7 cell line variant and a subset of phospho-Akt-negative breast carcinomas

We have used a novel variant of the human oestrogen receptor (ER)-positive MCF-7 cell line, TMX2-28, as a model to study breast cancer. TMX2-28 cells show no detectable levels of mRNA or protein expression for the ER and express basal cytokeratins (CKs) 5, 14, and 17. cDNA microarray comparison between TMX2-28 and its parent cell line, MCF-7, identified 1402 differentially expressed transcripts, one of which was, phospholipase D1 (PLD1). Using real-time RT–PCR, we confirmed that PLD1 mRNA levels are 10-fold higher in TMX2-28 cells than in MCF-7 cells. We next examined PLD1 expression in human breast carcinomas. Phospholipase D1 mRNA levels were higher in breast tumours that expressed high-mRNA levels of basal CKs 5 and/or 17, but PLD1 mRNA levels were not significantly higher in ER-negative tumours. Phospholipase D1 protein was overexpressed in 10 of 42 (24%) breast tumours examined by IHC. Phospholipase D1 was overexpressed in 6 of 31 ER-positive tumours and 4 of 11 ER-negative tumours. Phospholipase D1 was overexpressed in three of the four tumours that showed high CK5/17 expression. Five PLD1-positive tumours were negative for phospho-Akt expression, but positive for phospho-mammalian target of rapamycin (mTOR) expression. The other five PLD1-positive breast tumours showed positive expression for phospho-Akt; however, only two of these cases were positive for phospho-mTOR. In this study, we report that PLD1 and phospho-mTOR are coexpressed in a subset of phospho-Akt-negative breast carcinomas.

The RhoA pathway mediates MMP-2 and MMP-9-independent invasive behavior in a triple-negative breast cancer cell line.

Breast cancer is a heterogeneous disease that varies in its biology and response to therapy. A foremost threat to patients is tumor invasion and metastasis, with the greatest risk among patients diagnosed with triple‐negative and/or basal‐like breast cancers. A greater understanding of the molecular mechanisms underlying cancer cell spreading is needed as 90% of cancer‐associated deaths result from metastasis. We previously demonstrated that the Tamoxifen‐selected, MCF‐7 derivative, TMX2‐28, lacks expression of estrogen receptor α (ERα) and is highly invasive, yet maintains an epithelial morphology. The present study was designed to further characterize TMX2‐28 cells and elucidate their invasion mechanism. We found that TMX2‐28 cells do not express human epidermal growth factor receptor 2 (HER2) and progesterone receptor (PR), in addition to lacking ERα, making the cells triple‐negative. We then determined that TMX2‐28 cells lack expression of active matrix metalloproteinases (MMPs)‐1, MMP‐2, MMP‐9, and other genes involved in epithelial–mesenchymal transition (EMT) suggesting that TMX2‐28 may not utilize mesenchymal invasion. In contrast, TMX2‐28 cells have high expression of Ras Homolog Gene Family Member, A (RhoA), a protein known to play a critical role in amoeboid invasion. Blocking RhoA activity with the RhoA pathway specific inhibitor H‐1152, or a RhoA specific siRNA, resulted in inhibition of invasive behavior. Collectively, these results suggest that TMX2‐28 breast cancer cells exploit a RhoA‐dependent, proteolytic‐independent invasion mechanism. Targeting the RhoA pathway in triple‐negative, basal‐like breast cancers that have a proteolytic‐independent invasion mechanism may provide therapeutic strategies for the treatment of patients with increased risk of metastasis. J. Cell. Biochem. 114: 1385–1394, 2013.

TOXAPHENE IS ANTIESTROGENIC IN A HUMAN BREAST-CANCER CELL ASSAY

Toxaphene is a complex mixture of chlorinated bornanes, bornenes, and bornadienes and was a heavily used insecticide in the United States until its use was restricted in 1982. There are conflicting reports regarding the potential for toxaphene to induce estrogenic responses in human and nonhuman animals. Due to the public concern over environmental estrogens, the estrogenicity of toxaphene was examined in a human breast-cancer cell assay, the MCF-7 focus assay, which is based on in vitro postconfluent cell proliferation and tissue restructuring. In this assay, 0.1-1 nM 17beta-estradiol (E2) produces maximum postconfluent proliferation and formation of multicellular nodules or foci. Toxaphene was also tested for its ability (1) to bind the estrogen receptor (ER) in a competitive binding assay using recombinant human ERalpha (rhER) and in a whole-cell competitive ER binding assay, and (2) to alter the catabolism of E2 in MCF-7 cell cultures. Results from the MCF-7 focus assay showed: (1) Toxaphene alone was not estrogenic between the concentrations of 0.5 nM and 10 microM, (2) toxaphene in binary combinations with chlordane, dieldrin, or endosulfan (alpha or beta) was not estrogenic, and (3) toxaphene was weakly antiestrogenic (it reduced the number of foci induced by 0.1 nM and 0.01 nM E2). Results from the competitive binding assays showed that (1) toxaphene alone did not bind rhER or ER in MCF-7 cells, and (2) toxaphene in binary combinations with other pesticides did not bind rhER. Results from the growth assay and radiometric analysis of E2 catabolism showed that (1) toxaphene did not alter the growth rate of MCF-7 cell cultures over 13 d, and (2) toxaphene did not alter the catabolism of E2. In conclusion, results from the MCF-7 focus assay demonstrate that toxaphene is weakly antiestrogenic rather than estrogenic.Toxaphene is a complex mixture of chlorinated bornanes, bornenes, and bornadienes and was a heavily used insecticide in the United States until its use was restricted in 1982. There are conflicting reports regarding the potential for toxaphene to induce estrogenic responses in human and nonhuman animals. Due to the public concern over environmental estrogens, the estrogenicity of toxaphene was examined in a human breast-cancer cell assay, the MCF-7 focus assay, which is based on in vitro postconfluent cell proliferation and tissue restructuring. In this assay, 0.1-1 n M 17 b estradiol (E) produces maximum postconfluent proliferation and formation of multicel2 lular nodules or foci. Toxaphene was also tested for its ability (1) to bind the estrogen receptor (ER) in a competitive binding assay using recombinant human ER a (rhER) and in a whole-cell competitive ER binding assay, and (2) to alter the catabolism of E 2 in MCF-7 cell cultures. Results from the MCF-7 focus assay showed: (1) Toxaphene alone was not estrogenic between the concentrations of 0.5 n M and 10 mu M, (2) toxaphene in binary combinations with chlordane, dieldrin, or endosulfan (a or b) was not estrogenic, and (3) toxaphene was weakly antiestrogenic (it reduced the number of foci induced by 0.1 n M and 0.01 n M E2). Results from the competitive binding assays showed that (1) toxaphene alone did not bind rhER or ER in MCF-7 cells, and (2) toxaphene in binary combinations with other pesticides did not bind rhER. Results from the growth assay and radiometric analysis of E2 catabolism showed that (1) toxaphene did not alter the growth rate of MCF-7 cell cultures over 13 d, and (2) toxaphene did not alter the catabolism of E2. In conclusion, results from the MCF-7 focus assay demonstrate that toxaphene is weakly antiestrogenic rather than estrogenic.

Increased promoter methylation in exfoliated breast epithelial cells in women with a previous breast biopsy

Accurately identifying women at increased risk of developing breast cancer will provide greater opportunity for early detection and prevention. DNA promoter methylation is a promising biomarker for assessing breast cancer risk. Breast milk contains large numbers of exfoliated epithelial cells that are ideal for methylation analyses. Exfoliated epithelial cells were isolated from the milk obtained from each breast of 134 women with a history of a non-proliferative benign breast biopsy (Biopsy Group). Promoter methylation of three tumor suppressor genes, RASSF1, SFRP1 and GSTP1, was assessed by pyrosequencing of bisulfite-modified DNA. Methylation scores from the milk of the 134 women in the Biopsy Group were compared to scores from 102 women for whom a breast biopsy was not a recruitment requirement (Reference Group). Mean methylation scores for RASSF1 and GSTP1 were significantly higher in the Biopsy than in the Reference Group. For all three genes the percentage of outlier scores was greater in the Biopsy than in the Reference Group but reached statistical significance only for GSTP1. A comparison between the biopsied and non-biopsied breasts of the Biopsy Group revealed higher mean methylation and a greater number of outlier scores in the biopsied breast for both SFRP1 and RASSF1, but not for GSTP1. This is the first evidence of CpG island methylation in tumor suppressor genes of women who may be at increased risk of developing breast cancer based on having had a prior breast biopsy.