Rhythmic cardiac contractions depend on the organized propagation of depolarizing and repolarizing wavefronts. Repolarization is spatially heterogeneous and depends largely on gradients of potassium currents. Gradient disruption in heart disease may underlie susceptibility to fatal arrhythmias, but it is not known how this gradient is established. We show that, in mice lacking the homeodomain transcription factor Irx5, the cardiac repolarization gradient is abolished due to increased Kv4.2 potassium-channel expression in endocardial myocardium, resulting in a selective increase of the major cardiac repolarization current, I(to,f), and increased susceptibility to arrhythmias. Myocardial Irx5 is expressed in a gradient opposite that of Kv4.2, and Irx5 represses Kv4.2 expression by recruiting mBop, a cardiac transcriptional repressor. Thus, an Irx5 repressor gradient negatively regulates potassium-channel-gene expression in the heart, forming an inverse I(to,f) gradient that ensures coordinated cardiac repolarization while also preventing arrhythmias.

The cytotoxicity and tumor-targeting properties of the anti-HER2/neu monoclonal antibody trastuzumab modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localization sequence ([NLS] italicized) of simian virus 40 large T-antigen and radiolabeled with 111 In were evaluated. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) for reaction with NLSpeptides and labeled with 111 In using diethylenetriaminepentaacetic acid (DTPA). The immunoreactivity of 111 In-NLS-trastuzumab was determined by its ability to displace the binding of trastuzumab to SK-BR-3 human breast cancer (BC) cells. Cellular uptake and nuclear localization were evaluated in SK-BR-3, MDA-MB-361, and MDA-MB-231 BC cells, expressing high, intermediate, or very low levels of HER2/neu, respectively, by cell fractionation and confocal microscopy. Biodistribution and nuclear uptake were compared in athymic mice bearing MDA-MB-361 xenografts. The cytotoxicity of 111 In-trastuzumab and 111 In-NLS-trastuzumab was studied by clonogenic assays, and DNA damage was assessed by probing for phosphorylated histone H2AX (gH2AX) foci. Results: The dissociation constant for binding of 111 In-NLS-trastuzumab to SK-BR-3 cells was reduced ,3-fold compared with that of 111 In-trastuzumab, demonstrating relatively preserved receptorbinding affinity. The receptor-mediated internalization of 111 Intrastuzumab in SK-BR-3, MDA-MB-361, and MDA-MB-231 cells increased significantly from 7.2% 6 0.9%, 1.3% 6 0.1%, and 0.2% 6 0.05% to 14.4% 6 1.8%, 6.3% 6 0.2%, and 0.9% 6 0.2% for 111 In-NLS-trastuzumab harboring 6 NLS-peptides, respectively. NLS-trastuzumab localized in the nuclei of BC cells, whereas unmodified trastuzumab remained surface-bound. Conjugation of 111 In-trastuzumab to NLS-peptides did not affect its tissue biodistribution but promoted specific nuclear uptake in MDA-MB-361 xenografts (2.4-2.9 %ID/g [percentage injected dose per gram] for 111 In-NLS-trastuzumab and 1.1 %ID/g for 111 In-trastuzumab). 111 In-NLS-trastuzumab was 5-and 2-fold more potent at killing SK-BR-3 and MDA-MB-361 cells than 111 In-trastuzumab, respectively, whereas toxicity toward MDA-MB-231 cells was minimal. 111 In-NLS-trastuzumab was 6-fold more effective at killing SK-BR-3 cells than unlabeled trastuzumab. The development of recombinant antibodies for cancer therapy has emerged as one of the most promising areas in oncology (1). Trastuzumab (Herceptin; Hoffmann-La Roche), in particular, is a humanized monoclonal antibody (mAb) directed against the human epidermal growth factor receptor-2 (HER2/neu), a transmembrane receptor tyrosine kinase that is overexpressed in 25%-30% of breast cancers (BCs) and distant metastases (2). Trastuzumab shows clinical activity in women with HER2/neu-overexpressing metastatic BC and exhibits synergistic antitumor effects when combined with paclitaxel or anthracyclines, achieving overall response rates of 40%-60% (2). Despite its effectiveness in combination regimens, the response rate...

Our goals in this study were to determine whether 111 In-trastuzumab coupled to peptides harboring nuclear localizing sequences (NLSs) could kill trastuzumab-resistant breast cancer cell lines through the emission of Auger electrons and whether the combination of radiosensitization with methotrexate (MTX) would augment the cytotoxicity of this radiopharmaceutical. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate for reaction with NLS peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with 111 In. HER2 expression was determined by Western blot and by radioligand binding assay using 111 In-trastuzumab in a panel of breast cancer cell lines, including SK-BR-3, MDA-MB-231 and its HER2-transfected subclone (231-H2N), and 2 trastuzumab-resistant variants (TrR1 and TrR2). Nuclear importation of 111 In-NLS-trastuzumab and 111 Intrastuzumab in breast cancer cells was measured by subcellular fractionation, and the clonogenic survival of these cells was determined after incubation with 111 In-NLS-trastuzumab, 111 Intrastuzumab, or trastuzumab (combined with or without MTX). Survival curves were analyzed according to the dose-response model, and the radiation-enhancement ratio was calculated from the survival curve parameters. Results: The expression of HER2 was highest in SK-BR-3 cells (12.6 · 10 5 receptors/cell), compared with 231-H2N and TrR1 cells (6.1 · 10 5 and 5.1 · 10 5 receptors/cell, respectively), and lowest in MDA-MB-231 and TrR2 cells (0.4 · 10 5 and 0.6 · 10 5 receptors/cell, respectively). NLS peptides increased the nuclear uptake of 111 In-trastuzumab in MDA-MB-231, 231-H2N, TrR1, and TrR2 cells from 0.1% 6 0.01%, 2.5% 6 0.2%, 2.8% 6 0.7%, and 0.5% 6 0.1% to 0.5% 6 0.1%, 4.6% 6 0.1%, 5.2% 6 0.6%, and 1.5% 6 0.2%, respectively. The cytotoxicity of 111 In-NLS-trastuzumab on breast cancer cells was directly correlated with the HER2 expression densities of the cells. On a molar concentration basis, the effective concentration required to kill 50% of 231-H2N and TrR1 cells for 111 In-NLS-trastuzumab was 9-to 12-fold lower than for 111 Intrastuzumab and 16-to 77-fold lower than for trastuzumab.MDA-MB-231 and TrR2 cells were less sensitive to 111 In-NLStrastuzumab or 111 In-trastuzumab, and both cell lines were completely insensitive to trastuzumab. The radiation-enhancement ratio induced by MTX for 231-H2N and TrR1 cells after exposure to 111 In-NLS-trastuzumab was 1.42 and 1.68, respectively. Conclusion: Targeted Auger electron radioimmunotherapy with 111 In-NLS-trastuzumab can overcome resistance to trastuzumab, and MTX can potently enhance the sensitivity of HER2-overexpressing breast cancer cells to the lethal Auger electrons emitted by this radiopharmaceutical.

Pertuzumab is a HER2 dimerization inhibitor that binds to an epitope unique from that of trastuzumab. Our objective was to determine whether SPECT with 111 In-diethylenetriaminepentaacetic acid-pertuzumab ( 111 In-DTPA-pertuzumab) could sensitively detect an early molecular response to trastuzumab manifested by HER2 downregulation and a later tumor response revealed by a decreased number of HER2-positive viable tumor cells. Methods: Changes in HER2 density in SKBr-3 and MDA-MB-361 BC cells exposed to trastuzumab (14 mg/mL) in vitro were measured by saturation binding assays using 111 In-DTPA-pertuzumab and by confocal immunofluorescence microscopy and flow cytometry with fluorescein isothiocyanate-labeled HER2/neu antibodies. Imaging of HER2 downregulation was studied in vivo in athymic mice with subcutaneous MDA-MB-361 tumors treated for 3 d with trastuzumab (4 mg/kg) or nonspecific human IgG (hIgG) or phosphate-buffered saline (PBS). Imaging of tumor response to trastuzumab was studied in mice bearing subcutaneous MDA-MB-361 xenografts treated with trastuzumab (4 mg/kg), followed by weekly doses of nonspecific hIgG or rituximab or PBS (2 mg/ kg). Mice were imaged on a micro-SPECT/CT system at 72 h after injection of 111 In-DTPA-pertuzumab. Tumor and normal-tissue biodistribution was determined. Results: 111 In-DTPA-pertuzumab saturation binding to SKBr-3 and MDA-MB-361 cells was significantly decreased at 72 h after exposure in vitro to trastuzumab (14 mg/mL), compared with untreated controls (62% 6 2%, P , 0.0001; 32% 6 9%, P , 0.0002, respectively). After 3 d of trastuzumab, in vivo tumor uptake of 111 In-DTPA-pertuzumab decreased 2-fold in trastuzumab-versus PBS-treated mice (13.5 6 2.6 percentage injected dose per gram [%ID/g] vs. 28.5 6 9.1 %ID/g, respectively; P , 0.05). There was also a 2-fold decreased tumor uptake in trastuzumab-versus PBS-treated mice by image volume-of-interest analysis (P 5 0.05), suggesting trastuzumab-mediated HER2 downregulation. After 3 wk of trastuzumab, tumor uptake of 111 In-DTPA-pertuzumab decreased 4.5-fold, compared with PBS-treated mice (7.6 6 0.4 vs. 34.6 6 9.9 %ID/g, respectively; P , 0.001); this decrease was associated with an almost-completed eradication of HER2-positive tumor cells determined immunohistochemically. Mol ecular imaging is a powerful new tool that has great potential for aiding in the optimal use of novel targeted cancer therapies by revealing the expression of target receptors in situ on lesions throughout the body; probing downstream treatment-induced molecular events, thus providing early mechanistic evidence of tumor response; and monitoring the prior existence or emergence of resistance pathways implicated in treatment failure (1). Trastuzumab (Herceptin; Roche Pharmaceuticals) is a humanized IgG 1 monoclonal antibody (mAb) approved for the treatment of early and advanced breast cancer (BC) which overexpresses the HER2 transmembrane tyrosine kinase (2,3). HER2 positivity is evaluated in a primary BC biopsy by immunohistochemical staining for HER...

111 In-nuclear localization sequence-trastuzumab is a radioimmunotherapeutic agent consisting of trastuzumab modified with NLS peptides (CGYGPKKKRKVGG) and labeled with the Auger electron emitter 111 In. Our objectives were to evaluate the tumor growth-inhibitory properties and normal-tissue toxicity of 111 In-NLS-trastuzumab in mice after intraperitoneal administration. Methods: The pharmacokinetics of 111 In-NLS-trastuzumab after intravenous (tail vein) or intraperitoneal injection in BALB/c mice were compared. Normal-tissue toxicity was determined in BALB/c mice at 2 wk after intraperitoneal injection of 3.7-18.5 MBq (4 mg/kg) of 111 In-NLS-trastuzumab by monitoring body weight, histopathologic examination of tissues, and hematology (white blood cell, platelet, red blood cell, and hemoglobin) or clinical biochemistry (alanine transaminase and creatinine) parameters. A no-observable-adverse-effect-level (NOAEL) dose was defined. Athymic mice bearing subcutaneous MDA-MB-361 or MDA-MB-231 human breast cancer xenografts (5.0 · 10 5 or 0.5 · 10 5 HER2/cell, respectively) were treated with a single NOAEL dose or 2 doses administered intraperitoneally and separated by 2 wk. Control groups were administered 111 In-trastuzumab, trastuzumab, nonspecific 111 In-NLS-human IgG (hIgG), or normal saline. Results: The bioavailability of 111 In-NLS-trastuzumab after intraperitoneal injection was 0.7. The NOAEL dose was 9.25 MBq (4 mg/kg); doses greater than or equal to 18.5 MBq decreased white blood cell or platelet counts, and doses of 27.7 MBq decreased red blood cell counts. There was no increase in alanine transaminase or creatinine at any doses tested. There were no morphologic changes to the liver, kidneys, heart, or spleen or loss of body weight.

Obesity and metabolic comorbidities represent increasing health problems. Endocrine disrupting compounds (EDCs) are exogenous agents that change endocrine function and cause adverse health effects. Most EDCs are synthetic chemicals; some are natural food components as phytoestrogens. People are exposed to complex mixtures of chemicals throughout their lives. EDCs impact hormone-dependent metabolic systems and brain function. Laboratory and human studies provide compelling evidence that human chemical contamination can play a role in obesity epidemic. Chemical exposures may increase the risk of obesity by altering the differentiation of adipocytes. EDCs can alter methylation patterns and normal epigenetic programming in cells. Oxidative stress may be induced by many of these chemicals, and accumulating evidence indicates that it plays important roles in the etiology of chronic diseases. The individual sensitivity to chemicals is variable, depending on environment and ability to metabolize hazardous chemicals. A number of genes, especially those representing antioxidant and detoxification pathways, have potential application as biomarkers of risk assessment. The potential health effects of combined exposures make the risk assessment process more complex compared to the assessment of single chemicals. Techniques and methods need to be further developed to fill data gaps and increase the knowledge on harmful exposure combinations.

BackgroundObstructive sleep apnea (OSA) and metabolic syndrome, both closely related to obesity, often coexist in affected individuals; however, body mass index is not an accurate indicator of body fat and thus is not a good predictor of OSA and other comorbidities. The aim of this study was to investigate whether the occurrence of OSA could be associated with an altered body fat distribution and a more evident cardio metabolic risk independently from obesity and metabolic syndrome.Methods and Results171 consecutive patients (58 men and 113 women) were included in the study and underwent overnight polysomnography. Anthropometric data, blood pressure, lipid profile, glycaemic parameters were recorded. Body composition by DXA, two-dimensional echocardiography and carotid intima/media thickness measurement were performed. 67 patients (39.2%) had no OSA and 104 (60.8%) had OSA. The percentage of patients with metabolic syndrome was significantly higher among OSA patients (65.4%) that were older, heavier and showed a bigger and fatter heart compared to the control group. Upper body fat deposition index , the ratio between upper body fat (head, arms and trunk fat in kilograms) and lower body fat (legs fat in kilograms), was significantly increased in the OSA patients and significantly related to epicardial fat thickness. In patients with metabolic syndrome, multivariate regression analyses showed that upper body fat deposition index and epicardial fat showed the best association with OSA.ConclusionThe occurrence of OSA in obese people is more closely related to cardiac adiposity and to abnormal fat distribution rather than to the absolute amount of adipose tissue. In patients with metabolic syndrome the severity of OSA is associated with increase in left ventricular mass and carotid intima/media thickness.

Our goal in this study was to elucidate the mechanisms by which methotrexate radiosensitizes HER2-positive human breast cancer cells to the Auger electron emitter 111 In-trastuzumab modified with nuclear-localization sequence peptides ( 111 In-NLS-trastuzumab) and to compare these mechanisms with the potential sensitizing effects of paclitaxel and doxorubicin when combined with this radiopharmaceutical. Methods: Experiments were performed in MDA-MB-231 human breast cancer cells, their HER2-transfected subclones (231-H2N), and 2 trastuzumabresistant variants (trastuzumab-resistant-1 and -2 [TrR1 and TrR2]). Effects of coexposure of these cells to 111 In-NLS-trastuzumab and low-dose, radiosensitizing methotrexate, paclitaxel, or doxorubicin were assessed by clonogenic cell-survival assay. Quantification of residual DNA damage was measured by the gH2AX-immunofluorescence assay, and cell cycle distribution was measured by fluorescence-activated cell sorting analysis. The radiation-enhancement ratio was calculated as the ratio of the surviving fraction (SF) of cells treated with 111 In-NLStrastuzumab alone to that of cells treated concurrently with 111 In-NLS-trastuzumab and methotrexate, paclitaxel, or doxorubicin. Results: A reduction in the SF in HER2-positive 231-H2N (55.7% 6 1.3%) and TrR1 (62.6% 6 6.5%) cells was demonstrated after exposure to 111 In-NLS-trastuzumab (;0.2 MBq/mg, 100 nmol/L) but not in MDA-MB-231 or TrR2 cells expressing low levels of HER2 (SF . 90%, P . 0.05). Coadministration of methotrexate, paclitaxel, or doxorubicin enhanced the cytotoxicity of 111 In-NLS-trastuzumab toward 231-H2N and TrR1 cells but not toward MDA-MB-231 or TrR2 cells. The radiationenhancement ratios for methotrexate, paclitaxel, and doxorubicin for 231-H2N or TrR1 cells were 2.0-2.2, 1.6-1.8, and 2.7-2.8, respectively. Methotrexate or doxorubicin combined with 111 In-NLS-trastuzumab, compared to treatment with 111 In-NLStrastuzumab alone, significantly increased residual gH2AX foci in 231-H2N and TrR1 cells but not in MDA-MB-231 or TrR2 cells or in any cell line treated concurrently with paclitaxel and 111 In-NLS-trastuzumab. Cells exposed to low-dose methotrexate accumulated in the G 1 /S phase of the cell cycle, whereas low-dose paclitaxel or doxorubicin caused cells to arrest in the G 2 /M phase. Conclusion: Low-dose methotrexate, paclitaxel, or doxorubicin potently sensitized HER2-overexpressing human breast cancer cells, with and without acquired trastuzumab-resistance, to the Auger electron emissions from 111 In-NLS-trastuzumab through cell cycle distribution changes and in part through the inhibitory effects of these agents on DNA damage repair.