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1.  IRP2 regulates breast tumor growth 
Cancer research  2013;74(2):497-507.
Experimental and epidemiological evidence suggest that dysregulation of proteins involved in iron metabolism plays a critical role in cancer. The mechanisms by which cancer cells alter homeostatic iron regulation are just beginning to be understood. Here we demonstrate that iron regulatory protein 2 (IRP2) plays a key role in iron accumulation in breast cancer. Although both IRP1 and IRP2 are over-expressed in breast cancer, the overexpression of IRP2, but not IRP1, is associated with decreased ferritin H and increased transferrin receptor 1 (TfR1). Knock-down of IRP2 in triple negative MDA-MB-231 human breast cancer cells increases ferritin H expression and decreases TfR1 expression, resulting in a decrease in the labile iron pool. Further, IRP2 knockdown reduces growth of MDA-MB-231 cells in the mouse mammary fat pad. Gene expression microarray profiles of breast cancer patients demonstrate that increased IRP2 expression is associated with high grade cancer. Increased IRP2 expression is observed in luminal A, luminal B and basal breast cancer subtypes, but not in breast tumors of the ERBB2 molecular subtype. These results suggest that dysregulation of IRP2 is an early nodal point underlying altered iron metabolism in breast cancer and may contribute to poor outcome of some breast cancer patients.
doi:10.1158/0008-5472.CAN-13-1224
PMCID: PMC3989290  PMID: 24285726
breast cancer; iron; metabolism; iron regulatory proteins; molecular subtypes
2.  Epidemiological associations between iron and cardiovascular disease and diabetes 
Disruptions in iron homeostasis are linked to a broad spectrum of chronic conditions including cardiovascular, malignant, metabolic, and neurodegenerative disease. Evidence supporting this contention derives from a variety of analytical approaches, ranging from molecular to population-based studies. This review focuses on key epidemiological studies that assess the relationship between body iron status and chronic diseases, with particular emphasis on atherosclerosis ,metabolic syndrome and diabetes. Multiple surrogates have been used to measure body iron status, including serum ferritin, transferrin saturation, serum iron, and dietary iron intake. The lack of a uniform and standardized means of assessing body iron status has limited the precision of epidemiological associations. Intervention studies using depletion of iron to alter risk have been conducted. Genetic and molecular techniques have helped to explicate the biochemistry of iron metabolism at the molecular level. Plausible explanations for how iron contributes to the pathogenesis of these chronic diseases are beginning to be elucidated. Most evidence supports the hypothesis that excess iron contributes to chronic disease by fostering excess production of free radicals. Overall, epidemiological studies, reinforced by basic science experiments, provide a strong line of evidence supporting the association between iron and elevated risk of cardiovascular disease and diabetes. In this narrative review we attempt to condense the information from existing literature on this topic.
doi:10.3389/fphar.2014.00117
PMCID: PMC4033158  PMID: 24904420
iron; cardiovascular disease; diabetes mellitus; metabolic syndrome; epidemiologic studies
3.  Cellular iron metabolism in prognosis and therapy of breast cancer 
Critical reviews in oncogenesis  2013;18(5):435-448.
Despite many recent advances, breast cancer remains a clinical challenge. Current issues include improving prognostic evaluation and increasing therapeutic options for women whose tumors are refractory to current frontline therapies. Iron metabolism is frequently disrupted in breast cancer, and may offer an opportunity to address these challenges. Iron enhances breast tumor initiation, growth and metastases. Iron may contribute to breast tumor initiation by promoting redox cycling of estrogen metabolites. Up-regulation of iron import and down-regulation of iron export may enable breast cancer cells to acquire and retain excess iron. Alterations in iron metabolism in macrophages and other cells of the tumor microenvironment may also foster breast tumor growth. Expression of iron metabolic genes in breast tumors is predictive of breast cancer prognosis. Iron chelators and other strategies designed to limit iron may have therapeutic value in breast cancer. The dependence of breast cancer on iron presents rich opportunities for improved prognostic evaluation and therapeutic intervention.
PMCID: PMC3736347  PMID: 23879588
iron; breast cancer; estrogen
4.  Targeting Cancer Stem Cells with Nanoparticle-Enabled Therapies 
Journal of molecular biomarkers & diagnosis  2012;Suppl 8:10.4172/2155-9929.S8-003.
Emerging evidence suggests that multiple tumor types are sustained by a small population of transformed stem-like cells that have the ability to both self-renew and give rise to non-tumorigenic daughter cells that constitute the bulk of a tumor. These cells, which generally constitute a minority of the overall cancer cell population, are highly resistant to conventional therapies and persist following treatment, leading to disease relapse and the formation of distant metastases. Therapies that disrupt the maintenance and survival of cancer stem cells are the subject of active current investigation. This review discusses recent approaches to the application of nanomedicine to the targeting and elimination of cancer stem cells. Specifically, recent publications in the areas of nanoparticle-enabled drug and nucleic acid delivery and photothermal therapy are addressed.
doi:10.4172/2155-9929.S8-003
PMCID: PMC3875221  PMID: 24383043
Cancer stem cells; Tumor-initiating cells; Drug delivery; Nucleic acid delivery; Targeted; Nanoparticle; Carbon nanotube; Photo thermal therapy; Hyperthermia
5.  Serum Ferritin: Past, Present and Future 
Biochimica et biophysica acta  2010;1800(8):760-769.
Background
Serum ferritin was discovered in the 1930’s, and was developed as a clinical test in the 1970’s. Many diseases are associated with iron overload or iron deficiency. Serum ferritin is widely used in diagnosing and monitoring these diseases.
Scope of Review
In this chapter, we discuss the role of serum ferritin in physiological and pathological processes and its use as a clinical tool.
Major Conclusions
Although many aspects of the fundamental biology of serum ferritin remain surprisingly unclear, a growing number of roles have been attributed to extracellular ferritin, including newly described roles in iron delivery, angiogenesis, inflammation, immunity, signaling and cancer.
General Significance
Serum ferritin remains a clinically useful tool. Further studies on the biology of this protein may provide new biological insights.
doi:10.1016/j.bbagen.2010.03.011
PMCID: PMC2893236  PMID: 20304033
6.  FERRITIN H INDUCTION BY HISTONE DEACETYLASE INHIBITORS 
Biochemical pharmacology  2010;80(3):316-324.
Because both iron deficiency and iron excess are deleterious to normal cell function, the intracellular level of iron must be tightly controlled. Ferritin, an iron binding protein, regulates iron balance by storing iron in a bioavailable but non-toxic form. Ferritin protein comprises two subunits: ferritin H, which contains ferroxidase activity, and ferritin L. Here we demonstrate that ferritin H mRNA and protein are induced by histone deacetylase inhibitors (HDAC inhibitors), a promising class of anti-cancer drugs, in cultured human cancer cells. Deletion analysis and EMSA assays reveal that the induction of ferritin H occurs at a transcriptional level via Sp1 and NF-Y binding sites near the transcriptional start site of the human ferritin H promoter. Classically, HDAC inhibitors modulate gene expression by increasing histone acetylation. However, ChIP assays demonstrate that HDAC inhibitors induce ferritin H transcription by increasing NF-Y binding to the ferritin H promoter without changes in histone acetylation. These results identify ferritin H as a new target of HDAC inhibitors, and recruitment of NF-Y as a novel mechanism of action of HDAC inhibitors.
doi:10.1016/j.bcp.2010.04.008
PMCID: PMC2913600  PMID: 20385107
Ferritin H; histone acetylation; chromatin immunoprecipitation; cancer; HDAC inhibitors; transcription
7.  A Human Bone Morphogenetic Protein Antagonist Is Down-Regulated in Renal Cancer 
Molecular Biology of the Cell  2008;19(2):457-464.
We analyzed expression of candidate genes encoding cell surface or secreted proteins in normal kidney and kidney cancer. This screen identified a bone morphogenetic protein (BMP) antagonist, SOSTDC1 (sclerostin domain–containing-1) as down-regulated in kidney tumors. To confirm screening results, we probed cDNA dot blots with SOSTDC1. The SOSTDC1 message was decreased in 20/20 kidney tumors compared with normal kidney tissue. Immunohistochemistry confirmed significant decrease of SOSTDC1 protein in clear cell renal carcinomas relative to normal proximal renal tubule cells (p < 0.001). Expression of SOSTDC1 was not decreased in papillary and chromophobe kidney tumors. SOSTDC1 was abundantly expressed in podocytes, distal tubules, and transitional epithelia of the normal kidney. Transfection experiments demonstrated that SOSTDC1 is secreted and binds to neighboring cells and/or the extracellular matrix. SOSTDC1 suppresses both BMP-7–induced phosphorylation of R-Smads-1, -5, and -8 and Wnt-3a signaling. Restoration of SOSTDC1 in renal clear carcinoma cells profoundly suppresses proliferation. Collectively, these results demonstrate that SOSTDC1 is expressed in the human kidney and decreased in renal clear cell carcinoma. Because SOSTDC1 suppresses proliferation of renal carcinoma cells, restoration of SOSTDC1 signaling may represent a novel target in treatment of renal clear cell carcinoma.
doi:10.1091/mbc.E07-05-0433
PMCID: PMC2230586  PMID: 18032587
8.  Carbon nanotubes in hyperthermia therapy 
Advanced drug delivery reviews  2013;65(15):2045-2060.
Thermal tumor ablation therapies are being developed with a variety of nanomaterials, including single-and multiwalled carbon nanotubes. Carbon nanotubes (CNTs) have attracted interest due to their potential for simultaneous imaging and therapy. In this review, we highlight in vivo applications of carbon nanotube-mediated thermal therapy (CNMTT) and examine the rationale for use of this treatment in recurrent tumors or those resistant to conventional cancer therapies. Additionally, we discuss strategies to localize and enhance the cancer selectivity of this treatment and briefly examine issues relating the toxicity and long term fate of CNTs.
doi:10.1016/j.addr.2013.08.001
PMCID: PMC3914717  PMID: 23933617
photothermal therapy; carbon nanotubes; single-walled nanotubes; multiwalled nanotubes; cancer therapy
9.  Fluorinated Graphene Oxide; a New Multimodal Material for Biological Applications 
Fluorinated graphene oxide (FGO) is reported for the first time as a magnetically responsive drug carrier that can serve as a MRI and photoacoustic contrast agent, under pre-clinical settings, as well as a photothermal therapy Its hydrophilic nature facilitates biocompatibility. FGO as a broad wavelength absorber, with high charge transfer and strong nonlinear scattering is optimal for NIR laser-induced hyperthermia.
doi:10.1002/adma201301804
PMCID: PMC3938113  PMID: 24038195
multifunctional graphene; MRI; photoacoustic; photothermal therapy; drug delivery
10.  Iron and cancer: more ore to be mined 
Nature reviews. Cancer  2013;13(5):342-355.
Iron is an essential nutrient that facilitates cell proliferation and growth. However, iron also has the capacity to engage in redox cycling and free radical formation. Therefore, iron can contribute to both tumour initiation and tumour growth; recent work has also shown that iron has a role in the tumour microenvironment and in metastasis. Pathways of iron acquisition, efflux, storage and regulation are all perturbed in cancer, suggesting that reprogramming of iron metabolism is a central aspect of tumour cell survival. Signalling through hypoxia-inducible factor (HIF) and WNT pathways may contribute to altered iron metabolism in cancer. Targeting iron metabolic pathways may provide new tools for cancer prognosis and therapy.
doi:10.1038/nrc3495
PMCID: PMC4036554  PMID: 23594855
11.  Synthetic and natural iron chelators: therapeutic potential and clinical use 
Future medicinal chemistry  2009;1(9):10.4155/fmc.09.121.
Iron-chelation therapy has its origins in the treatment of iron-overload syndromes. For many years, the standard for this purpose has been deferoxamine. Recently, considerable progress has been made in identifying synthetic chelators with improved pharmacologic properties relative to deferoxamine. Most notable are deferasirox (Exjade®) and deferiprone (Ferriprox®), which are now available clinically. In addition to treatment of iron overload, there is an emerging role for iron chelators in the treatment of diseases characterized by oxidative stress, including cardiovascular disease, atherosclerosis, neurodegenerative diseases and cancer. While iron is not regarded as the underlying cause of these diseases, it does play an important role in disease progression, either through promotion of cellular growth and proliferation or through participation in redox reactions that catalyze the formation of reactive oxygen species and increase oxidative stress. Thus, iron chelators may be of therapeutic benefit in many of these conditions. Phytochemicals, many of which bind iron, may also owe some of their beneficial properties to iron chelation. This review will focus on the advances in iron-chelation therapy for the treatment of iron-overload disease and cancer, as well as neurodegenerative and chronic inflammatory diseases. Established and novel iron chelators will be discussed, as well as the emerging role of dietary plant polyphenols that effectively modulate iron biochemistry.
doi:10.4155/fmc.09.121
PMCID: PMC3821171  PMID: 21425984
12.  Ferroportin and Iron Regulation in Breast Cancer Progression and Prognosis 
Science translational medicine  2010;2(43):43ra56.
Ferroportin and hepcidin are critical proteins for the regulation of systemic iron homeostasis. Ferroportin is the only known mechanism for export of intracellular non–heme-associated iron; its stability is regulated by the hormone hepcidin. Although ferroportin profoundly affects concentrations of intracellular iron in tissues important for systemic iron absorption and trafficking, ferroportin concentrations in breast cancer and their influence on growth and prognosis have not been examined. We demonstrate here that both ferroportin and hepcidin are expressed in cultured human breast epithelial cells and that hepcidin regulates ferroportin in these cells. Further, ferroportin protein is substantially reduced in breast cancer cells compared to nonmalignant breast epithelial cells; ferroportin protein abundance correlates with metabolically available iron. Ferroportin protein is also present in normal human mammary tissue and markedly decreased in breast cancer tissue, with the highest degree of anaplasia associated with lowest ferroportin expression. Transfection of breast cancer cells with ferroportin significantly reduces their growth after orthotopic implantation in the mouse mammary fat pad. Gene expression profiles in breast cancers from >800 women reveal that decreased ferroportin gene expression is associated with a significant reduction in metastasis-free and disease-specific survival that is independent of other breast cancer risk factors. High ferroportin and low hepcidin gene expression identifies an extremely favorable cohort of breast cancer patients who have a 10-year survival of >90%. Ferroportin is a pivotal protein in breast biology and a strong and independent predictor of prognosis in breast cancer.
doi:10.1126/scisignal.3001127
PMCID: PMC3734848  PMID: 20686179
13.  Photothermal Response of Human and Murine Cancer Cells to Multiwalled Carbon Nanotubes after Laser Irradiation 
Cancer research  2010;70(23):9855-9864.
This study demonstrates the capability of multiwalled carbon nanotubes (MWNTs) coupled with laser irradiation to enhance treatment of cancer cells through enhanced and more controlled thermal deposition, increased tumor injury, and diminished heat shock protein (HSP) expression. We also explored the potential promise of MWNTs as drug delivery agents by observing the degree of intracellular uptake of these nanoparticles. To determine the heat generation capability of MWNTs, the absorption spectra and temperature rise during heating were measured. Higher optical absorption was observed for MWNTs in water compared with water alone. For identical laser parameters, MWNT-containing samples produced a significantly greater temperature elevation compared to samples treated with laser alone. Human prostate cancer (PC3) and murine renal carcinoma (RENCA) cells were irradiated with a 1,064-nm laser with an irradiance of 15.3 W/cm2 for 2 heating durations (1.5 and 5 minutes) alone or in combination with MWNT inclusion. Cytotoxicity and HSP expression following laser heating was used to determine the efficacy of laser treatment alone or in combination with MWNTs. No toxicity was observed for MWNTs alone. Inclusion of MWNTs dramatically decreased cell viability and HSP expression when combined with laser irradiation. MWNT cell internalization was measured using fluorescence and transmission electron microscopy following incubation of MWNTs with cells. With increasing incubation duration, a greater number of MWNTs were observed in cellular vacuoles and nuclei. These findings offer an initial proof of concept for the application of MWNTs in cancer therapy.
doi:10.1158/0008-5472.CAN-10-0250
PMCID: PMC3699181  PMID: 21098701
14.  SOSTDC1 differentially modulates Smad and beta-catenin activation and is down-regulated in breast cancer 
Sclerostin domain containing 1 (SOSTDC1) protein regulates processes from development to cancer by modulating activity of bone morphogenetic protein (BMP) and wingless/int (Wnt) signaling pathways. As dysregulation of both BMP and Wnt signaling has been observed in breast cancer, we investigated whether disruption of SOSTDC1 signaling occurs in breast cancer. SOSTDC1 mRNA expression levels in breast tissue were examined using a dot blot. Affymetrix microarray data on SOSTDC1 levels were correlated with breast cancer patient survival using Kaplan–Meier plots. Correlations between SOSTDC1 protein levels and clinical parameters were assessed by immunohistochemistry of a breast cancer tissue microarray. SOSTDC1 secretion and BMP and Wnt signaling were investigated using immunoblotting. We found that SOSTDC1 is expressed in normal breast tissue and this expression is reduced in breast cancer. High levels of SOSTDC1 mRNA correlated with increased patient survival; conversely, SOSTDC1 protein levels decreased as tumor size and disease stage increased. Treatment of breast cancer cells with recombinant SOSTDC1 or Wise, a SOSTDC1 orthologue, demonstrated that SOSTDC1 selectively blocks BMP-7-induced Smad phosphorylation without diminishing BMP-2 or Wnt3a-induced signaling. In conclusion, SOSTDC1 mRNA and protein are reduced in breast cancer. High SOSTDC1 mRNA levels correlate with increased distant metastasis-free survival in breast cancer patients. SOSTDC1 differentially affects Wnt3a, BMP-2, and BMP-7 signaling in breast cancer cells. These results identify SOSTDC1 as a clinically important extracellular regulator of multiple signaling pathways in breast cancer.
doi:10.1007/s10549-010-1261-9
PMCID: PMC3685185  PMID: 21113658
Beta-catenin (BMP); Bone morphogenetic protein(BMP); Breast cancer; Sclerostin domain containing 1 (SOSTDC1); Wingless/int (Wnt); Wise
15.  Hybrid 2D Nanomaterials as Dual-mode Contrast Agents in Cellular Imaging 
doi:10.1002/adma.201200706
PMCID: PMC3395317  PMID: 22573478
hybrid materials; imaging techniques; magnetic resonance imaging; luminescence; graphene oxide
16.  Application of circuit simulation method for differential modeling of TIM-2 iron uptake and metabolism in mouse kidney cells 
Circuit simulation is a powerful methodology to generate differential mathematical models. Due to its highly accurate modeling capability, circuit simulation can be used to investigate interactions between the parts and processes of a cellular system. Circuit simulation has become a core technology for the field of electrical engineering, but its application in biology has not yet been fully realized. As a case study for evaluating the more advanced features of a circuit simulation tool called Advanced Design System (ADS), we collected and modeled laboratory data for iron metabolism in mouse kidney cells for a H ferritin (HFt) receptor, T cell immunoglobulin and mucin domain-2 (TIM-2). The internal controlling parameters of TIM-2 associated iron metabolism were extracted and the ratios of iron movement among cellular compartments were quantified by ADS. The differential model processed by circuit simulation demonstrated a capability to identify variables and predict outcomes that could not be readily measured by in vitro experiments. For example, an initial rate of uptake of iron-loaded HFt (Fe-HFt) was 2.17 pmol per million cells. TIM-2 binding probability with Fe-HFt was 16.6%. An average of 8.5 min was required for the complex of TIM-2 and Fe-HFt to form an endosome. The endosome containing HFt lasted roughly 2 h. At the end of endocytosis, about 28% HFt remained intact and the rest was degraded. Iron released from degraded HFt was in the labile iron pool (LIP) and stimulated the generation of endogenous HFt for new storage. Both experimental data and the model showed that TIM-2 was not involved in the process of iron export. The extracted internal controlling parameters successfully captured the complexity of TIM-2 pathway and the use of circuit simulation-based modeling across a wider range of cellular systems is the next step for validating the significance and utility of this method.
doi:10.3389/fphys.2013.00136
PMCID: PMC3675319  PMID: 23761763
circuit simulator; export; ferritin; iron; model; storage; TIM-2; uptake
17.  The Resistance of Breast Cancer Stem Cells to Conventional Hyperthermia and their Sensitivity to Nanoparticle-mediated Photothermal Therapy 
Biomaterials  2012;33(10):2961-2970.
Breast tumors contain a small population of tumor initiating stem-like cells, termed breast cancer stem cells (BCSCs). These cells, which are refractory to chemotherapy and radiotherapy, are thought to persist following treatment and drive tumor recurrence. We examined whether BCSCs are similarly resistant to hyperthermic therapy, and whether nanoparticles could be used to overcome this resistance. Using a model of triple-negative breast cancer stem cells, we show that BCSCs are markedly resistant to traditional hyperthermia and become enriched in the surviving cell population following treatment. In contrast, BCSCs are sensitive to nanotube-mediated thermal treatment and lose their long-term proliferative capacity after nanotube-mediated thermal therapy. Moreover, use of this therapy in vivo promotes complete tumor regression and long-term survival of mice bearing cancer stem cell-driven breast tumors. Mechanistically, nanotube thermal therapy promotes rapid membrane permeabilization and necrosis of BCSCs. These data suggest that nanotube-mediated thermal treatment can simultaneously eliminate both the differentiated cells that constitute the bulk of a tumor and the BCSCs that drive tumor growth and recurrence.
doi:10.1016/j.biomaterials.2011.12.052
PMCID: PMC3286596  PMID: 22245557
18.  An Iron Regulatory Gene Signature Predicts Outcome in Breast Cancer 
Cancer research  2011;71(21):6728-6737.
Changes in iron regulation characterize the malignant state. However, the pathways that effect these changes and their specific impact on prognosis remain poorly understood. We capitalized on publicly available microarray datasets comprising 674 breast cancer cases to systematically investigate how expression of genes related to iron metabolism is linked to breast cancer prognosis. Of 61 genes involved in iron regulation, 49% were statistically significantly associated with distant metastasis-free survival (DMFS). Cases were divided into test and training cohorts and the supervised principal component method was used to stratify cases into risk groups. Optimal risk stratification was achieved with a model comprising 16 genes, which we term the iron regulatory gene signature (IRGS). Multivariable analysis revealed that the IRGS contributes information not captured by conventional prognostic indicators (hazard ratio 1.61; 95% CI 1.16–2.24; p=0.004). The IRGS successfully stratified homogeneously treated patients, including ER+ patients treated with tamoxifen monotherapy, both with (p=0.006) and without (p=0.03) lymph node metastases. To test whether multiple pathways were embedded within the IRGS, we evaluated the performance of two gene dyads with known roles in iron biology in ER+ patients treated with tamoxifen monotherapy (n=371). For both dyads, gene combinations that minimized intracellular iron content (anti-import: TFRCLow/HFEHigh; or pro-export: FPHigh/HAMPLow) were associated with favorable prognosis (p<0.005). Although the clinical utility of the IRGS will require further evaluation, its ability to both identify high risk patients within traditionally low risk groups and low risk patients within high risk groups has the potential to affect therapeutic decision-making.
doi:10.1158/0008-5472.CAN-11-1870
PMCID: PMC3206152  PMID: 21875943
19.  Determinants of the Thrombogenic Potential of Multiwalled Carbon Nanotubes 
Biomaterials  2011;32(26):5970-5978.
Multiwalled carbon nanotubes (MWCNTs) are cylindrical tubes of graphitic carbon with unique physical and electrical properties. MWCNTs are being explored for a variety of diagnostic and therapeutic applications. Successful biomedical application of MWCNTs will require compatibility with normal circulatory components, including constituents of the hemostatic cascades. In this manuscript, we compare the thrombotic activity of MWCNTs in vitro and in vivo. We also assess the influence of functionalization of MWCNTs on thrombotic activity. In vitro, MWCNT activate the intrinsic pathway of coagulation as measured by activated partial thromboplastin time (aPTT) assays. Functionalization by amidation or carboxylation enhances this procoagulant activity. Mechanistic studies demonstrate that MWCNTs enhance propagation of the intrinsic pathway via a non-classical mechanism strongly dependent on factor IX. MWCNTs preferentially associate with factor IXa and may provide a platform for its activation. In addition to their effects on the coagulation cascade, MWCNTs activate platelets in vitro, with amidated MWCNTs exhibiting greater platelet activation than carboxylated or pristine MWCNTs. However, contrasting trends are obtained in vivo, where functionalization tends to diminish rather than enhance pro-coagulant activity. Thus, following systemic injection of MWCNTs in mice, pristine MWCNTs decreased platelet counts, increased vWF, and increased D-dimers. In contrast, carboxylated MWCNTS exhibited little procoagulant tendency in vivo, eliciting only a mild and transient decrease in platelets. Amidated MWCNTs elicited no statistically significant change in platelet count. Further, neither carboxylated nor amidated MWCNTs increased vWF or D-dimers in mouse plasma. We conclude that the pro-coagulant tendencies of MWCNTs observed in vitro are not necessarily recapitulated in vivo. Further, functionalization can markedly attenuate the procoagulant activity of MWCNTs in vivo. This work will inform the rational development of biocompatible MWCNTs for systemic delivery.
doi:10.1016/j.biomaterials.2011.04.059
PMCID: PMC3130101  PMID: 21663954
blood; blood compatibility; clotting; nanoparticle; platelet activation; thrombosis
20.  Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells 
PLoS ONE  2012;7(7):e40030.
Angiogenesis is tightly regulated through complex crosstalk between pro- and anti-angiogenic signals. High molecular weight kininogen (HK) is an endogenous protein that is proteolytically cleaved in plasma and on endothelial cell surfaces to HKa, an anti-angiogenic protein. Ferritin binds to HKa and blocks its anti-angiogenic activity. Here, we explore mechanisms underlying the cytoprotective effect of ferritin in endothelial cells exposed to HKa. We observe that ferritin promotes adhesion and survival of HKa-treated cells and restores key survival and adhesion signaling pathways mediated by Erk, Akt, FAK and paxillin. We further elucidate structural motifs of both HKa and ferritin that are required for effects on endothelial cells. We identify an histidine-glycine-lysine (HGK) -rich antiproliferative region within domain 5 of HK as the target of ferritin, and demonstrate that both ferritin subunits of the H and L type regulate HKa activity. We further demonstrate that ferritin reduces binding of HKa to endothelial cells and restores the association of uPAR with α5β1 integrin. We propose that ferritin blocks the anti-angiogenic activity of HKa by reducing binding of HKa to UPAR and interfering with anti-adhesive and anti-proliferative signaling of HKa.
doi:10.1371/journal.pone.0040030
PMCID: PMC3388046  PMID: 22768328
21.  Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress 
Free radical biology & medicine  2011;50(11):1565-1574.
Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMEC), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5), were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrates that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. While normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMEC cells under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. This study discusses some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from the response to acute oxidative stress in normal mammary epithelial cells.
doi:10.1016/j.freeradbiomed.2011.03.002
PMCID: PMC3119600  PMID: 21397008
Oxidative stress; breast cancer; human mammary epithelial cells; microarrays; glucose oxidase; GluOx
22.  Ironing Out Cancer 
Cancer research  2011;71(5):1511-1514.
New insights into the roles of proteins that regulate cellular iron in cancer growth, angiogenesis, and metastasis have recently emerged. Discoveries of the roles of ferroportin, hepcidin, Lcn2, and members of the STEAP and IRP families in cancer have provided specificity and molecular definition to the role of iron homeostasis in cancer growth and metastasis. A number of studies directly support a role of these proteins in modifying bio-available iron, while other studies suggest that at least some of their effects are independent of their role in iron biology.
doi:10.1158/0008-5472.CAN-10-3614
PMCID: PMC3079335  PMID: 21363917
23.  Development of iron-containing multiwalled carbon nanotubes for MR-guided laser-induced thermotherapy 
Nanomedicine (London, England)  2011;6(8):1341-1352.
Aims
To test iron-containing multiwalled carbon nanotubes (MWCNTs) as bifunctional nanomaterials for imaging and thermal ablation of tumors.
Materials & Methods
MWCNTs entrapping iron were synthesized by chemical vapor deposition. The T2-weighted contrast enhancement properties of MWCNTs containing increasing amounts of iron were determined in vitro. Suspensions of these particles were injected into tumor-bearing mice and tracked longitudinally over 7 days by MRI. Heat-generating abilities of these nanomaterials following exposure to near infrared (NIR) laser irradiation was determined in vitro and in vivo.
Results
The magnetic resonance contrast properties of carbon nanotubes were directly related to their iron content. Iron-containing nanotubes were functional T2-weighted contrast agents in vitro and could be imaged in vivo long-term following injection. Iron content of nanotubes did not affect their ability to generate thermoablative temperatures following exposure to NIR and significant tumor regression was observed in mice treated with MWCNTs and NIR laser irradiation.
Conclusion
These data demonstrate that iron-containing MWCNTs are functional T2-weighted contrast agents and efficient mediators of tumor-specific thermal ablation in vivo.
doi:10.2217/nnm.11.37
PMCID: PMC3197747  PMID: 21506687
cancer; contrast agent; in vivo; laser; MRI; nanotube; T2; thermal therapy
24.  Iron Uptake Mediated by Binding of H-Ferritin to the TIM-2 Receptor in Mouse Cells 
PLoS ONE  2011;6(8):e23800.
Ferritin binds specifically and saturably to a variety of cell types, and recently several ferritin receptors have been cloned. TIM-2 is a specific receptor for H ferritin (HFt) in the mouse. TIM-2 is a member of the T cell immunoglobulin and mucin domain containing (TIM) protein family and plays an important role in immunity. The expression of TIM-2 outside of the immune system indicates that this receptor may have broader roles. We tested whether ferritin binding to TIM-2 can serve as an iron delivery mechanism. TIM-2 was transfected into normal (TCMK-1) mouse kidney cells, where it was appropriately expressed on the cell surface. HFt was labeled with 55Fe and 55Fe-HFt was incubated with TIM-2 positive cells or controls. 55Fe-HFt uptake was observed only in TIM-2 positive cells. HFt uptake was also seen in A20 B cells, which express endogenous TIM-2. TIM-2 levels were not increased by iron chelation. Uptake of 55Fe-HFt was specific and temperature-dependent. HFt taken up by TIM-2 positive cells transited through the endosome and eventually entered a lysosomal compartment, distinguishing the HFt pathway from that of transferrin, the classical vehicle for cellular iron delivery. Iron delivered following binding of HFt to TIM-2 entered the cytosol and became metabolically available, resulting in increased levels of endogenous intracellular ferritin. We conclude that TIM-2 can function as an iron uptake pathway.
doi:10.1371/journal.pone.0023800
PMCID: PMC3158792  PMID: 21886823
25.  Treatment of breast cancer through the application of irreversible electroporation using a novel minimally invasive single needle electrode 
Irreversible electroporation (IRE) is a therapeutic technology for the ablation of soft tissues using electrodes to deliver intense but short electric pulses across a cell membrane, creating nanopores that lead to cell death. This phenomenon only affects the cell membrane, leaving the extracellular matrix and sensitive structures intact, making it a promising technique for the treatment many types of tumors. In this paper, we present the first in vivo study to achieve tumor regression using a translatable, clinically relevant single needle electrode for treatment administration. Numerical models of the electric field distribution for the protocol used suggest that a 1000 V/cm field threshold is sufficient to treat a tumor, and that the electric field distribution will slightly decrease if the same protocol were used on a tumor deep seated within a human breast. Tumor regression was observed in 5 out of 7 MDA-MB231 human mammary tumors orthotopically implanted in female Nu/Nu mice, with continued growth in controls.
doi:10.1007/s10549-010-0803-5
PMCID: PMC3021965  PMID: 20191380
Tumor ablation; IRE; N-TIRE; Minimally invasive surgery; Electropermeabilization; Focal orthotopic tumor therapy; Translational protocols

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