2-2--(hydroxynitrosohydrazono)bis-ethanamine and Breast-Neoplasms

In response to the need for reliable cellular models that reflect complex tumor microenvironmental properties, and enable more precise testing of anti-cancer therapeutics effects on humans, a co-culture platform for in-vitro model that enhances the physiology of breast cancer (BC) microenvironment is presented. A six well imaging plate wherein each macro-well contains several separate compartments was designed. Three-dimensional (3D) cancer spheroids are generated and cultured in the inner compartment which is embossed with an array of nano-liter micro-chambers made of hydrogel. Stromal cells are cultured in the outer chambers. The two cell types are cultured side-by-side, sharing a common space, thus enabling extra-cellular communication via secreted molecules. As proof of concept, a model of BC tumor microenvironment was recapitulated by co-cultivating 3D MCF7 spheroids in the presence of tumor-associated macrophages (TAMs). The presence of TAMs induced an aggressive phenotype by promoting spheroid growth, enhancing survivin expression levels and enabling invasive behavior. Moreover, TAMs influenced the response of BC spheroids to cytotoxic treatment as well as hormonal drug therapy, and enhanced the effects of nitric oxide donor. The platform enables time-lapse imaging and treatment without losing spatial location of the measured spheroids, thereby allowing measurements and analysis at individual-object resolution in an easy and efficient manner.

Topics: Antineoplastic Agents; Breast Neoplasms; Coculture Techniques; Doxorubicin; Drug Evaluation, Preclinical; Humans; Hydrogels; Macrophages; MCF-7 Cells; Models, Biological; Spheroids, Cellular; Stromal Cells; Tamoxifen; Triazenes; Tumor Microenvironment; U937 Cells

Vascular endothelial dysfunction and platelet activation play a key role in tumor metastasis, and therefore, both of these processes are considered important therapeutic targets in cancer. The aim of our studies was to analyze antimetastatic activity of combination therapy using nitric oxide donor DETA/NO and antiplatelet drug clopidogrel. Nitric oxide acts as a vasoprotective mediator, while clopidogrel inhibits ADP-mediated platelet aggregation. 4T1-luc2-tdTomato cell line transplanted intravenously (i.v.) and 4T1 cell line transplanted orthotopically were used as metastatic mammary gland cancer models. Moreover, antiaggregation action of compounds was tested ex vivo on the blood samples taken from breast cancer patients. We have shown that in selected dosage regimes, DETA/NO combined with clopidogrel significantly reduced lung metastatic foci formation in an i.v. model, and such inhibition was transiently observed also in an orthotopic model. The antimetastatic effect was correlated with a significant increase of prostacyclin (PGI2) metabolite and reduction of endothelin-1, sE-selectin, sI-CAM, and TGF-β plasma levels as well as decreased V-CAM expression on the endothelium. Combination therapy decreased fibrinogen binding to the resting platelets at the early stage of tumor progression (day 14). However, at the later stages (days 21 and 28), the markers of platelet activation were detected (increased JON/A antibody bound, P-selectin level, binding of fibrinogen, and vWf). Decreased aggregation as well as a lower release of TGF-β were detected in platelets incubated ex vivo with compounds tested from metastatic breast cancer patients. Although combination therapy increases E-cadherin, the increase of N-cadherin and α-SMA in tumor tissue was also observed. The results showed that at the early stages of tumor progression, combined therapy with DETA/NO and clopidogrel improves vasoprotective and antiplatelet activity. However, in advanced tumors, some adverse effects toward platelet activation can be observed.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Clopidogrel; Endothelium, Vascular; Female; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Nitric Oxide Donors; Platelet Activation; Platelet Aggregation Inhibitors; Platelet-Rich Plasma; Triazenes

We have previously demonstrated that relatively high concentrations of NO [Nitric Oxide] as produced by activated macrophages induced apoptosis in the human breast cancer cell line, MDA-MB-468. More recently, we also demonstrated the importance of endogenous H2O2 in the regulation of growth in human breast cancer cells. In the present study we assessed the interplay between exogenously administered NO and the endogenously produced reactive oxygen species [ROS] in human breast cancer cells and evaluated the mechanism[s] in the induction of apoptosis. To this end we identified a novel mechanism by which NO down regulated endogenous hydrogen peroxide [H2O2] formation via the down-regulation of superoxide [O2 (.-)] and the activation of catalase. We further demonstrated the existence of a feed forward mechanistic loop involving protein phosphatase 2A [PP2A] and its downstream substrate FOXO1 in the induction of apoptosis and the synthesis of catalase. We utilized gene silencing of PP2A, FOXO1 and catalase to assess their relative importance and key roles in NO mediated apoptosis. This study provides the potential for a therapeutic approach in treating breast cancer by targeted delivery of NO where NO donors and activators of downstream players could initiate a self sustaining apoptotic cascade in breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Catalase; Cell Line, Tumor; Female; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Inorganic Pyrophosphatase; Mitochondrial Proteins; Nitric Oxide; Nitroso Compounds; Reactive Oxygen Species; Superoxides

Nitric oxide (NO), a free radical, has been implicated in the biology of human cancers, including breast cancer, yet it is still unclear how NO affects tumor development and propagation. We herein gradually adapted four human breast adenocarcinoma cell lines (BT-20, Hs578T, T-47D, and MCF-7) to increasing concentrations of the NO donor DETA-NONOate up to 600 muM. The resulting model system consisted of a set of fully adapted high nitric oxide ("HNO") cell lines that are biologically different from the "parent" cell lines from which they originated. Although each of the four parent and HNO cell lines had identical morphologic appearance, the HNO cells grew faster than their corresponding parent cells and were resistant to both nitrogen- and oxygen-based free radicals. These cell lines serve as a novel tool to study the role of NO in breast cancer progression and potentially can be used to predict the therapeutic response leading to more efficient therapeutic regimens.

Topics: Adaptation, Physiological; Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Humans; Hydrogen Peroxide; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds

Nitric oxide (NO) in nanomolar (nmol/L) concentrations is consistently detected in tumor microenvironment and has been found to promote tumorigenesis. The mechanism by which NO enhances tumor progression is largely unknown. In this study, we investigated the possible mechanisms and identified cellular targets by which NO increases proliferation of human breast cancer cell lines MDA-MB-231 and MCF-7. DETA-NONOate, a long acting NO donor, with a half-life of 20 h, was used. We found that NO (nmol/L) dramatically increased total protein synthesis in MDA-MB-231 and MCF-7 and also increased cell proliferation. NO specifically increased the translation of cyclin D1 and ornithine decarboxylase (ODC) without altering their mRNA levels or half-lives. Critical components in the translational machinery, such as phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets, phosphorylated eukaryotic translation initiation factor and p70 S6 kinase, were up-regulated following NO treatment, and inhibition of mTOR with rapamycin attenuated NO induced increase of cyclin D1 and ODC. Activation of translational machinery was mediated by NO-induced up-regulation of the Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase/ERK (Raf/MEK/ERK) and phosphatidylinositol 3-kinase (PI-3 kinase)/Akt signaling pathways. Up-regulation of the Raf/MEK/ERK and PI-3 kinase/Akt pathways by NO was found to be mediated by activation of Ras, which was cyclic guanosine 3',5'-monophosphate independent. Furthermore, inactivation of Ras by farnesyl transferase inhibitor or K-Ras small interfering RNA attenuated NO-induced increase in proliferation signaling and cyclin D1 and ODC translation, further confirming the involvement of Ras activation during NO-induced cell proliferation.

Topics: Alkyl and Aryl Transferases; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cyclic GMP; Dose-Response Relationship, Drug; Eukaryotic Initiation Factor-4E; Humans; MAP Kinase Signaling System; Nitric Oxide; Nitroso Compounds; Phosphatidylinositol 3-Kinases; Protein Biosynthesis; Protein Kinases; Proto-Oncogene Proteins c-akt; raf Kinases; TOR Serine-Threonine Kinases

We have previously reported that nitric oxide (NO) induces apoptosis in MDA-MB-468 cells through its action on the mitochondria and the release of cytochrome c. In this study, we investigated the critical events that must occur after which these cells are committed to apoptosis. We used the long-acting NO donor DETA-NONOate, which, at a concentration of 1 mM, releases NO in the range produced by activated macrophages. Depolarization of mitochondrial membrane potential (MMP) occurred at 4 h of DETA-NONOate treatment, which returned to control values and which was followed by another wave of depolarization at 24 h. There was a 2-fold increase of cytochrome c in the cytosol at 6 h, but it was not until 36 h that the level of cytochrome c was increased by 15-fold. Although the initial release of cytochrome c from the mitochondria could be inhibited by cyclosporin A or by bongkrekic acid, the later release continued even in its presence. We observed that the later release of cytochrome c at 36 h was independent of MMP depolarization but was dependent on Bax integration into the mitochondrial membrane, which committed the cells to apoptosis. We also observed a decline in the levels of cytosolic phospho-Akt at 16-24 h of DETA-NONOate treatment. We also conclude that decrease in phospho-Akt is an essential event upstream from Bax integration in MDA-MB-468 cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cytochrome c Group; Cytosol; Humans; Hydrazines; Intracellular Membranes; Membrane Potentials; Mitochondria; Nitric Oxide Donors; Nitroso Compounds; Oligonucleotides, Antisense; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Apoptosis is regulated by a series of biochemical events that commits a cell to death. We are interested in understanding and have been investigating the mechanisms by which nitric oxide (NO) induces apoptosis in human breast cancer cell lines. In this study, we investigated the possible interplay of extracellular signal-regulated kinase (ERK) and Akt pathways in NO-induced apoptosis. MKP-1 transcripts were induced in these cells as early as 4 h, peaking at 8 h leading to inactivation of ERK1/2 at 16-24 h after exposure to NO. We also found 50% decrease in the levels pAkt at 24 h of DETA-NONOate treatment. The inactivation of ERK1/2 preceded the dephosphorylation of Akt and apoptosis. NO was not able to inactivate ERK1/2 or Akt or to induce apoptosis in the presence of a phosphatase inhibitor, sodium orthovanadate, or antisense oligonucleotides, suggesting a cross-talk between the two pathways. NO also up-regulated MKP-1 in another breast cancer cell line, ZR 75-30, which led to inactivation of ERK1/2 and induced apoptosis. In MDA-MB-231, NO did not induce MKP-1, and there was no ERK inactivation or apoptosis. Our results indicate that expression of MKP-1 by NO leading to dephosphorylation of ERK1/2 is the initial essential event that commits the cells to the apoptotic pathway in breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Dual Specificity Phosphatase 1; Enzyme Induction; Humans; Immediate-Early Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G(1) phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 microM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G(1) phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G(1) phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G(1) phase of the cell cycle.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cyclin D1; Down-Regulation; G1 Phase; Growth Inhibitors; Humans; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Tumor Cells, Cultured

High amounts of nitric oxide (NO) produced by activated macrophages or NO donors are required to induce cytotoxicity and apoptosis in pathogens and tumor cells. High concentrations of NO may lead to nonspecific toxicity thereby limiting the use of NO donors in the treatment of cancer. In this study, we tested the possibility of potentiating the apoptotic action of NO in a human breast cancer cell line, MDA-MB-468, by combining it with a farnesyltransferase inhibitor (FTI), which has been shown to induce apoptosis in some other cancer cell lines with minimal toxicity to normal cells. DETA-NONOate, a long acting NO donor which has a half-life of 20 h at 37 degrees C, was used in this study. DETA-NONOate (1 mM), which releases NO in the range produced by activated macrophages, induced apoptosis after 36 h in MDA-MB-468 cells via cytochrome c release and caspase-9 and -3 activation. FTI (25 microM) potentiated the action of lower concentrations of DETA-NONOate (25-100 microM) by inducing apoptosis in these cells within 24 h by increasing cytochrome c release and caspase-9 and -3 activation. This effect was observed preferentially in the cancer cell lines studied with no apoptosis induction in normal breast epithelial cells. This novel combination of FTI and NO may emerge as a promising approach for the treatment of breast cancer.

Topics: Alkyl and Aryl Transferases; Apoptosis; Breast Neoplasms; Drug Synergism; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Tumor Cells, Cultured